Hereditary Hormone Excess: Genes, Molecular Pathways, and Syndromes

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1 X/05/$20.00/0 Endocrine Reviews 26(5): Printed in U.S.A. Copyright 2005 by The Endocrine Society doi: /er Hereditary Hormone Excess: Genes, Molecular Pathways, and Syndromes Stephen J. Marx and William F. Simonds Metabolic Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland Hereditary origin of a tumor helps toward early discovery of its mutated gene; for example, it supports the compilation of a DNA panel from index cases to identify that gene by finding mutations in it. The gene for a hereditary tumor may contribute also to common tumors. For some syndromes, such as hereditary paraganglioma, several genes can cause a similar syndrome. For other syndromes, such as multiple endocrine neoplasia 2, one gene supports variants of a syndrome. Onset usually begins earlier and in more locations with hereditary than sporadic tumors. Mono- or oligoclonal ( clonal ) tumor usually implies a postnatal delay, albeit less delay than for sporadic tumor, to onset and potential for cancer. Hormone excess from a polyclonal tissue shows onset at birth and no benefit from subtotal ablation of the secreting organ. Genes can cause neoplasms through stepwise loss of function, gain of function, or combinations of these. Polyclonal hormonal excess reflects abnormal gene dosage or effect, such as activation or haploinsufficiency. Polyclonal hyperplasia can I. Introduction: Frameworks for Organizing Syndromes of Hormonal Excess A. Older frameworks B. Current frameworks and syndrome coverage II. Heredity Contributes to Gene Identification A. Broad routes to identification of a gene that is tumorigenic in the germline B. Heredity in a tumor syndrome supports two independent mapping techniques for narrowing the subchromosomal interval of candidate genes C. Panel of germline DNAs from several index cases often holds several different mutations in one sought disease gene: sequencing that gene in the panel and finding the mutations is a powerful criterion for disease gene identification III. The Gene for a Hereditary Syndrome Often Contributes to Sporadic Tumors A. One mutation can have similar expressions in hereditary or sporadic tumors B. One mutation can have different expressions in hereditary or sporadic tumors IV. Hereditary Hormonal Excess Syndromes, Genes, and Molecules A. Hereditary monoclonal excess limited to one hyperfunctioning hormonal tissue B. Hereditary hormonal excess in one monoclonal tissue within a multiple neoplasia syndrome C. Multiple endocrine neoplasia (MEN) syndromes D. Mosaic states E. Hereditary syndromes with polyclonal hyperfunction of one hormonal tissue cause the main endpoint of clinical expression in some syndromes or can be a precursor to clonal progression in others. Gene discovery is usually the first step toward clarifying the molecule and pathway mutated in a syndrome. Most mutated pathways in hormone excess states are only partly understood. The bases for tissue specificity of hormone excess syndromes are usually uncertain. In a few syndromes, tissue selectivity arises from mutation in the open reading frame of a regulatory gene (CASR, TSHR) with selective expression driven by its promoter. Polyclonal excess of a hormone is usually from a defect in the sensor system for an extracellular ligand (e.g., calcium, glucose, TSH). The final connections of any of these polyclonal or clonal pathways to hormone secretion have not been identified. In many cases, monoclonal proliferation causes hormone excess, probably as a secondary consequence of accumulation of cells with coincidental hormone-secretory ability. (Endocrine Reviews 26: , 2005) V. Relations of Monoclonal and Polyclonal Components of Hyperfunction A. Steps in monoclonal evolution B. Polyclonal features within syndromes of hormonal excess C. Steps in histopathology different from or beyond hyperplasia D. Relations of tumorigenic germline mutation to sporadic tumor E. Animal models genes acting alone or in cooperation for tumorigenesis VI. Tissue Specificity in Hereditary Hormone Excess A. Expressions of tissue specificity in hormone excess syndromes B. Mechanisms for tissue specificity of hormonal excess syndromes VII. Syndrome and Gene Classification According to Mutated Process and Pathway A. Overview of downstream pathways and broad endpoints B. Hypoxia/angiogenesis pathways C. Cell cycle pathways D. Apoptosis pathways E. Housekeeping processes F. Genome stabilization pathways G. Signal transduction pathways and processes H. Genes contributing to monoclonal neoplasia by unknown pathway and processes I. Implications about relation between a mutated pathway and hormone excess VIII. Conclusion 615

2 616 Endocrine Reviews, August 2005, 26(5): Marx and Simonds Hereditary Tumors I. Introduction: Frameworks for Organizing Syndromes of Hormonal Excess HORMONE EXCESS IS the ultimate expression from diverse disorders; mutant genes are prominent causes. Their mutations may be germline, somatic, or a combination of both. Hereditary causes represent only a minority of all hormone excess states 1 but have a special place. For one thing, they can present special opportunities that make initial discovery of the mutated gene easier. For another, the gene identified for a rare hereditary syndrome of hormone excess often has broad implications in normal and abnormal states. Hereditary hormone excess is usually part of a primarily multiorgan syndrome. The grouping of affected tissues in a syndrome usually has not matched an intuitive or otherwise logical pattern. Several broad classification frameworks have been proposed to help deal with this concern. A. Older frameworks Recognition of familial neoplasms 2 was first reported in the 1800s (1) as tuberous sclerosis complex (TSC) and peripheral neurofibromatosis (NF) type 1 (NF1) (2 4). Obviously, a component of a syndrome could not have been understood as hormonal until after the major discoveries of hormones, which did not begin until the early 1900s. Heredity as a feature of hormonal excess was not well-recognized until its extended characterization in multiple endocrine neoplasia (MEN) type 1 (MEN1) in 1954 (5 7). Heredity in von Hippel-Lindau disease (VHL) was not established until a decade later (8). Although the concepts of heredity have been progressively strengthened, certain terms to organize these disorders have not proved durable. The category of phakomatosis was derived from the Greek phakos for birthmark; this term was developed originally to categorize syndromes with tumors of First Published Online January 4, 2005 Abbreviations: APC, Adenomatous polyposis of the colon; APUD, amine precursor uptake and decarboxylation; cas-r, calcium-sensing receptor; CDK, cyclin-dependent kinase; FAP, familial adenomatous polyposis; FHH, familial hypocalciuric or benign hypercalcemia; FIH, familial isolated hyperparathyroidism; FIPh, familial isolated pheochromocytoma; FMTC, familial isolated MTC; GAP, GTPase activating protein; GlnDH, glutamate dehydrogenase; G protein, guanine nucleotide regulatory protein; GPCR, G protein-coupled transmembrane receptor; Gs, stimulatory isoform of G protein; HIF, hypoxia-inducing factor; HPT-JT, hyperparathyroidism-jaw tumor syndrome; LH-R, LH receptor; LOH, loss of heterozygosity; LOI, loss of imprinting; MEN, multiple endocrine neoplasia; MTC, medullary thyroid cancer; NF, neurofibromatosis; NSHPT, neonatal severe primary hyperparathyroidism; PGL, paraganglioma; PHHI, persistent hyperinsulinemic hypoglycemia of infancy; PI3, phosphatidylinositol-3; RCC, renal cell carcinoma; sdh, succinate dehydrogenase; TSC, tuberous sclerosis complex; TSHR, TSH receptor; VEGF, vascular endothelial growth factor; VHL, von Hippel- Lindau disease. 1 Hormone secretion is abbreviated herein by hormonal or secretory. These terms do not refer herein to primary excess function in a hormone target tissue, such as breast cancer with BRCA1 mutation or the renal tubule in Liddle syndrome. 2 The terms neoplasia and tumor are used in the traditional sense, referring to new growth that may be monoclonal or polyclonal. Endocrine Reviews is published bimonthly by The Endocrine Society ( the foremost professional society serving the endocrine community. the central nervous system and skin, such as TSC and peripheral NF (9). The clinical similarities in these syndromes suggested that they had related causes. This classification remains in occasional use today, although with many modifications (10). An authoritative, current definition of phakomatosis illustrates its acquired complexity: conditions that predispose to hamartomas and other tumors and involve the skin and/or eyes, nervous system, and one or more body systems (11). No common cause among the phakomatoses has been recognized. The method of argentaffin or silver-based staining was initially developed in 1870 (12) and supported a histological classification method. Argentaffin staining, a rough correlate with neuroendocrine origin, was cited as a defining feature of several tumors, including several tumors in MEN1 and MEN2 (13). It was already evident in 1970 that tumors of neuroectoderm origin were not all positive for the argentaffin stain. The nonspecific nature of argentaffin methods derives from histochemical reactivity with selected small amines (14). Pearse (15, 16) and others promoted a related classification, based on histochemical and developmental concepts, designating certain tissues as APUD, based on their potential for amine precursor uptake and decarboxylation. Subsequently, this was expanded into the concept of a neuroendocrine system. The APUD classification has long been questioned by some embryologists as an oversimplification, which did not account for valid embryological origin of certain structures (17). At least one related concept has persisted. Certain tumors in MEN2 (C cell neoplasia and adrenal medullary neoplasia) are attributed to an origin from one neuroendocrine structure, specifically the neural crest (18, 19); however, the inherent parathyroid tumors seem to conflict with any suggestion of unitary origin of all MEN2A tumors from neural crest (17). Nesidioblastosis was proposed as the basis for pancreatic islet tumors (20). As with the APUD concept, this concept was based in histology and embryology. Nesidioblastosis is a normal embryonic process in which pancreatic ductules give rise to buds that could develop into islets. When pathologically increased, nesidioblastosis might be central in hypersecretion of insulin, other hormones, and other factors (21). Nesidioblastosis was considered as possibly contributing to two states that were incompletely separated: persistent hyperinsulinemic hypoglycemia of infancy (PHHI), and pancreatic islet macro- and microadenomas in adults with and without MEN1. More recent evaluations have found that nesidioblastosis is sometimes a feature of the normal pancreas at all ages. Also, it has not been confirmed as characteristic within any syndrome. Histopathological interpretation of abnormalities in islet hyperfunction syndromes has shifted to other features (22 24). Tumor multiplicity was recognized early in many neoplasia syndromes and has also supported concepts of humoral contributions to tumorigenesis. In particular, the islets or other tissues in MEN1 have been suggested to release factors that acted via the bloodstream to promote extrapancreatic hormonal tumors (25, 26). Limited support came from suggestions of gastrin acting as a trophic factor for gastric car-

3 Marx and Simonds Hereditary Tumors Endocrine Reviews, August 2005, 26(5): TABLE 1. Disproportionate identification of mutated genes in hereditary vs. nonhereditary hyperfunction of hormonal tissues Tissue Hereditary fraction a cinoid tumors (27) and for a humoral role of epidermal growth factor-like peptides in NF1 (28). Lastly, GH-secreting tumors promote distant release of IGF-I, which arguably exerts its own tumorigenic effects (29). The other hypothesized, humoral factors in the multiple neoplasias have not been fully identified. Subsequent evidence, however, favored a more central role for intrinsic defects, such as multifocal independent monoclonal expansions, 3 as the major explanation for tumor multiplicity (30 32). B. Current frameworks and syndrome coverage Current frameworks for states of hereditary hormone excess are more anatomical and more etiologically neutral than several earlier ones. Terms like isolated hyperparathyroidism, neuroendocrine, enteropancreatic, and multiple neoplasia are typical. Similarly, the continued use of eponyms for a few syndromes is noncommittal about etiology. At an increasing rate, the molecular basis of many syndromes is being clarified. Important aspects have been reviewed recently (33 36). This paper covers the following topics about hereditary hormonal excess: processes of tumorigenesis, mutated genes, their encoded defective molecules, disturbed pathways, and tissue patterns of expression (particularly hormonal expressions). A major focus of this paper is the hormone secretory (see Footnote 1) aspects of certain syndromes. To include relevant exceptions, this coverage includes hereditary syndromes with gland overgrowth and an often unrealized potential for hormone oversecretion, such as thyroid cancer, carcinoid tumor, and other so-called nonfunctional hormonal tumors (islet, pituitary, adrenal cortex). Conditions mimicking hormone excess but arising from mutation in hormone target tissues are not included in this review. For example, Liddle syndrome is omitted; this can arise from activating mutation in either of two subunits of the epithelial 3 The term monoclonal herein implies monoclonality or oligoclonality that has been proved or judged highly likely. The term polyclonal implies polyclonality that is highly likely. Identified mutated genes Hereditary or mosaic cause Nonhereditary cause Parathyroid 0.05 MEN1, b RET, CASR, HRPT2 b PTH-CYCD1 (231) Chromaffin 0.30 RET, b VHL, b SDHD, b SDHC, SDHB, NF1, MEN1 P53 (445) Adrenal (corticosteroids) 0.02 MEN1, PRKAR1A, P53, b GNAS b HRAS, NRAS (446) Adrenal/Leydig (androgens) 0.02 LHR, b GNAS, P53, b PRKAR1A HRAS, NRAS Gastrin cells 0.25 MEN1 b DPC4/SMAD4 (447) Islet -cells 0.10 MEN1, b VHL, TSC2, SUR1, Kir6.2, GK, GLUD1 None Somatostatin cells 0.10 NF1 None Islet (nonfunctional) 0.10 MEN1, b VHL, TSC2 None Anterior pituitary 0.15 MEN1, b PRKAR1A, GNAS b GNAI2 (448) (any hormone or none) Foregut carcinoid 0.10 MEN1 b -Catenin (449) Hindgut carcinoid 0.01 None P53 (450) Thyrocyte (non C cell) 0.05 PTEN, PRKAR1A, WRN, APC, GNAS, TSHR b RET-PTC, -Catenin, P53, TRs, BRAF ( ) Thyroid C cells 0.10 RET b None a Hereditary fraction for one tissue type of tumor is estimated from multiple sources. No definitive population surveys of this parameter exist for any of the tissues. b Some of the genes causing hereditary excess may also cause nonhereditary excess; these genes are represented only under the hereditary category. sodium channel, mimicking but not associated with hyperaldosteronism (37). Similarly, tissue hyperfunction resulting from upstream regulation, such as ACTH action with steroidogenesis defects, is not covered. II. Heredity Contributes to Gene Identification The majority of cases with hormone excess are not hereditary. The best estimates of the hereditary component vary from 0% among hindgut carcinoids and 5% among parathyroid tumors or among nonmedullary thyroid tumors (38, 39) to greater than 25% among pheochromocytomas and among gastrinomas (31, 40) (Table 1). Furthermore, the familial basis of a tumor may be usually obvious as in gastrinoma or usually occult as in pheochromocytoma (40, 41). Heredity, per se, facilitates identification of a gene that predisposes to a rare syndrome of hormonal excess. Stated differently, there has been a lower fraction of gene identification among the far more common, nonhereditary hormonal tumors. The presence of many as yet undiscovered tumorigenic genes with somatic mutation in sporadic tumors is suggested by subchromosomal and even submicroscopic loci of gain or loss of alleles in many hormonal tumors (42, 43). This apparent paradox of more ready identification of some of the genes mutated in rare hereditary tumors has several explanations, based on the mechanisms of tumorigenesis and, consequently, the methods developed for identification of tumorigenic genes. A. Broad routes to identification of a gene that is tumorigenic in the germline 1. Purification of a biological activity associated with a protein or a cdna a. Functional cloning. Identification of a disease gene before the middle 1980s relied mainly on functional cloning (44): gene derivation after biochemical purification of a protein

4 618 Endocrine Reviews, August 2005, 26(5): Marx and Simonds Hereditary Tumors associated with a special biological function for example, a pigment (hemoglobin), a peptide hormone, or an enzyme. b. Expression cloning. In expression cloning, a detectable specific biological activity, such as that from the calciumsensing receptor (cas-r), in a pool of many cdnas, was expressed and measured in transfected reporter cells; then, the activity was purified by serial enrichment, expression, and repeated selection among cdna pools (45). Functional cloning or expression cloning was never possible for identification of most tumorigenic genes, because an underlying biochemical disturbance could not be predicted or recognized and then purified before identification of the gene. However, selected tumorigenic genes were identified through their broad oncogenic properties that they could transmit to susceptible reporter cells. In particular, mouse NIH-3T3 cells were exploited for their ability to be transformed by so-called direct acting oncogenes. These methods allowed the identification of RET, MET, RAS, RAF, SRC, SIS, MYC, FOS, JUN, and a limited number of other oncogenes (46). Many oncogenes have been implicated to varying degrees in sporadic tumors. Few (such as RET, MET, and CDK4) have been implicated in hereditary tumors. Although some oncogenes could thus be isolated in susceptible cells, no equivalent system has been developed for identifying the many tumor suppressor genes. Their shared tumor suppressor property would inhibit cell accumulation and, thereby, impair enrichment by cell accumulation. 2. Precise subchromosomal mapping of the disease trait a. Positional cloning. The first, generally applicable, mapping approach was positional cloning (47). In positional cloning, the subchromosomal interval of candidate genes is narrowed as much as possible to allow the subsequent steps; these are the cloning of all or most genes in the narrowed candidate gene interval and then sequencing among these to find the defining mutations in a panel of germline DNAs from index cases. b. Positional candidate approach. An alternative approach has been the positional candidate approach (47). First, the subchromosomal interval of candidate genes is narrowed as above. Because some or even all genes in the candidate interval had already been characterized in part, those that seem most promising as candidates are tested selectively for mutations. Both of these approaches became more powerful as the map of the human genome approached completion. Still, only about half of unidentified disease genes can be predicted from appealing candidate genes. The remainder have uninformative sequence (48). If the positional candidate approach is not successful, positional cloning remains as the default method. B. Heredity in a tumor syndrome supports two independent mapping techniques for narrowing the subchromosomal interval of candidate genes 1. Reconstruction of meiotic recombination events from haplotypes. Haplotype analysis in families permits reconstruction and analysis of an archival meiotic crossover (same as meiotic recombination) event in development of a gamete. A meiotic crossingover event on the gamete carrying the mutated candidate gene can establish, near the candidate gene locus, a boundary in the chromosomal map of that gamete. All markers on one side of the boundary segregate with the disease phenotype. All markers on the other side of the boundary are traceable to an unaffected ancestor and thus define a zone of excluded candidate genes. A candidate interval can be narrowed by retrospective analysis of haplotype maps of many gametes to reconstruct the progressively rare crossing-over, closer and closer to the candidate gene. Such methods can narrow a candidate interval to one million bases (about 1 cm). An analogous evaluation can be done among multiple families with the same syndrome. If several families are discovered to share a statistically significant and diseaseassociated core haplotype including the candidate gene locus, it generally means that they inherited that haplotype with the associated disease-predisposing mutation from a common ancestor with that mutation (49). This core haplotype, then, becomes conceptually similar to boundaries from meioses from crossing-over events in a single extended kindred. 2. Tumoral loss of heterozygosity about the locus of candidate genes. The first step in tumorigenesis is often a single base DNA change or other small mutation of one allele within a tumorigenic gene; this mutation is present in every cell if it was acquired through germline transmission but may cause no phenotype in the subject or in the cell. If the gene contributes to tumor development by a loss-of-function mechanism, a second step in tumorigenesis is often a large subchromosomal or whole chromosomal rearrangement that removes the remaining normal copy of that same gene along with the contiguous copies of many genes on either side from the same DNA strand (32). This reduction to the null status of the syndromal gene frees one tumor precursor cell to proliferate into a tumor clone; the same large DNA rearrangement is thus present in all cells of the tumor clone. Similar biallelic events in one tumor precursor cell can lead to a hereditary or a nonhereditary tumor clone. The triggering rearrangement can be recognized from its outcome as a loss of alleles from the previously normal copy of the chromosome in the tumor DNA when a subchromosomal DNA marker is heterozygous in the germline (usually tested with leukocyte DNA as a germline surrogate); this is termed loss of heterozygosity (LOH) or allelic loss about the locus of that gene. If the tumor syndrome is hereditary, if multiple syndromic tumors are tested, and if there is consistent LOH at the presumed germline locus, then there is a high likelihood that the LOH of every tumor with LOH at this locus gives boundaries that bracket the sought gene. This method can be strengthened by haplotype analysis involving comparison of tumor DNA with other tumor DNA or with germline DNA of several family members to establish that allelic losses were from the chromosome of the unaffected parent (49). If a tumor is not hereditary, there must be uncertainty about the identity of the gene(s) with the first hit within that locus. A substantial fraction of all genes, through a loss of function, can contribute to or coexist with cell accumulation. If the initial copy with loss of function does not involve the gene

5 Marx and Simonds Hereditary Tumors Endocrine Reviews, August 2005, 26(5): of interest, then any derived boundary for LOH near that locus can give information that would be misleading for identification of that syndromal gene. C. Panel of germline DNAs from several index cases often holds several different mutations in one sought disease gene: sequencing that gene in the panel and finding the mutations is a powerful criterion for disease gene identification Obviously, the final step in identification of a disease gene is proof that mutation in one candidate gene causes the disease. The usual criterion is that mutation therein associates clearly with the rare and specific syndrome. Most of the genes described in this paper were identified through a combination of subchromosomal mapping and then identification by the demonstration of disease-associated mutations. When a family with a well-characterized rare syndrome is identified, there is high likelihood that the family has mutation in the same gene. Typically, a panel of germline DNAs is compiled with one index case from each of several such kindreds and with no suspicion of common ancestry, making it less likely that the same mutation is represented more than once. Although phenocopies of a syndrome sometimes exist based on mutation in other genes (such as with at least four different genes whose mutation causes PHHI; see Section IV.E.5.c), phenocopy can occasionally be excluded in a family by demonstrating genetic linkage to the candidate locus. Not surprisingly, there is at least one relevant exception to the specificity of this application of genetic linkage: SUR1 or Kir6.2 are not homologs, but either can be mutated in indistinguishable families with PHHI, and each maps to approximately the same location in chromosome 11p15 (see Section IV.E.5.e). III. The Gene for a Hereditary Syndrome Often Contributes to Sporadic Tumors A. One mutation can have similar expressions in hereditary or sporadic tumors 1. Tissue selectivity. The mutated gene and even the specific mutation within a gene may convey striking tissue selectivity to the tumor process (Table 1) (see Section VI.A). Of course, the specificity also pertains to the group of organs in which a given mutated gene can contribute to tumors. For example, the MEN1 gene contributes to either hereditary or sporadic tumors in the parathyroids, the pancreatic islets, bronchial carcinoid, and certain other tissues (31). Similarly, the RET gene contributes to hereditary tumor and to a lower fraction of sporadic tumors, in thyroidal C cells and adrenal medulla (50). 2. Degree of morbidity, particularly cancer and its aggressiveness. Aggressiveness, as manifested by cancer potential, earlier age at tumor onset, and/or embryonal lethality, is inherent in certain mutated genes and in selected mutations of certain genes. For example, loss of function of the hswi5/ini1 gene contributes to atypical teratoid/rhabdoid tumors of the central nervous system and several other nonhormonal tumors that are malignant in the first year of life in either sporadic or familial tumors (51, 52). Similar trends toward aggressive expressions are seen with a specific type of mutation in some hereditary or nonhereditary hormonal neoplasms. For example, the germline methionine-918 mutations in RET are associated with highly aggressive and early-onset medullary thyroid cancer (MTC) in the MEN2B syndrome. Among sporadic MTC, the identical mutation in somatic DNA is also associated with more aggressive tumor (53, 54) (see Sections IV.A.5 and IV.C.2). B. One mutation can have different expressions in hereditary or sporadic tumors 1. Tumor multiplicity in hereditary tumors. Unlike sporadic tumors, many hereditary tumors, particularly those with high penetrance, express multiplicity. In fact, lack of tumor multiplicity within an organ is unusual and puzzling in a highly penetrant hereditary tumor, such as the pituitary in MEN1 (55). Multiplicity is recognized by any one among several criteria. First, the term multiple describes tumors in differing organs within a syndrome such as the parathyroid, pancreatic islets, and the pituitary in MEN1 (31). Second, multiplicity describes tumor in separate portions of one tissue that normally is anatomically discontinuous. This multiplicity is sometimes striking in a dispersed organ such as the pancreatic islets, the duodenal endocrine tissues, or the parathyroids or in a paired organ such as the adrenal cortex or adrenal medulla (56). Third, tumors can be multiple even within a continuous tissue. When expressed as tumor nodules, this is readily recognizable as in the thyroid (57, 58). But when not nodular, this type of multiplicity may not be detected without use of special analyses, such as microdissection (56). 2. Earlier age at onset of hereditary tumors. For certain tumors, age of onset is earlier in the hereditary than the nonhereditary setting. The age differential may be striking, absent, or even reversed. For example, comparing tumors in MEN1 cases vs. sporadic cases, the difference in age of onset for parathyroid tumors is 30 yr (age 25 vs. 55 yr); for gastrinomas it is only 10 yr (age 35 vs. 45 yr), but for prolactinoma there is no age difference (age 35 yr in either) (31, 59, 60). This age differential can occur from tumor suppressor genes or oncogenes. For example, the approximate age of onset for palpable MTC for untreated cases with MEN2B (i.e., mainly mutated RET methionine-918 in the germline) vs. in sporadic cases (25% same mutation, somatically) is age 20 vs. 50 yr (61). This age differential is not seen for RET mutations that cause familial MTC (FMTC). 3. Differences in tissue selectivity. Certain genes or even certain mutations contribute to hereditary or sporadic tumors of the same tissue but in widely differing proportions. For example, RET germline mutation is associated with parathyroid tumor in 30 60% of cases with MEN2A, but no similar or other somatic mutation of the RET gene has so far been identified in a sporadic parathyroid tumor (62, 63). Similarly, MEN1 biallelic inactivation is implicated in most uterine fibroids of women with MEN1 but rarely if ever in sporadic uterine fibroid tumor (64). Conversely, somatic P53 mutation in

6 620 Endocrine Reviews, August 2005, 26(5): Marx and Simonds Hereditary Tumors serine-249 is frequent in hepatocellular cancer, likely reflecting direct consequences of aflatoxin exposure in somatic tissue (65); in contrast, liver cancer and particularly germline mutation of this codon are rare in Li-Fraumeni syndrome. 4. Fatal to the embryo. For certain genes, selected mutations, which contribute to sporadic tumors, are not seen in hereditary tumors, raising the possibility that, if that mutation occurred in the germline, it would be lethal during embryogenesis. This may apply to several tumorigenic mutations of some tumor suppressors and to most known mutations in oncogenes. For example, RAS mutation is found in half of colon cancers (sporadic or hereditary); however, RAS mutation has not been found to be transmitted through the germline (66, 67). IV. Hereditary Hormonal Excess Syndromes, Genes, and Molecules Syndromes are recognized when they have high penetrance and are consistent within and across families. Many mutations undoubtedly cause variable and weakly penetrant expressions (68, 69). Although these can be of great importance, including by contributing to multigenic phenotypes, few such mutations have been identified in man, and this topic is not covered further in this paper. A syndrome often is pathognomonic of germline mutation in just one or a very small number of alternate genes. This section of the paper reviews the pairing between syndromes, their mutated genes, and their mutant molecules. A later section covers relations between syndromes, their disturbed molecular pathways, and hormone excess. A. Hereditary monoclonal excess limited to one hyperfunctioning hormonal tissue 1. Isolated hyperparathyroidism a. Expressions. A syndrome of familial isolated hyperparathyroidism (FIH), unrelated to incomplete expression of another syndrome, has not been fully documented in a large kindred. In theory, FIH could reflect incomplete expression of any of four or more complex syndromes as follows: MEN1, MEN2A, familial hypocalciuric or benign hypercalcemia (FHH), or hyperparathyroidism-jaw tumor syndrome (HPT- JT) (70). In surveys of FIH, the etiologies of about one third are somewhat evenly divided between incomplete expressions of MEN1, FHH, or HPT-JT (70, 71). The remaining fraction of families with FIH could have other undiscovered syndromes or true FIH. Each of the two largest reported families with FIH and MEN1 mutation had 14 affected or unaffected carriers (72, 73). Based on a few large kindreds, there is a formal possibility that isolated hyperparathyroidism from mutation of MEN1 could be a durable phenotype. In analogy, another unusual and large family with CASR mutation but not expressing FHH has an FIH phenotype, with hypercalciuria, stones, monoclonal parathyroid adenomas, and benefit from subtotal parathyroidectomy (74, 75). b. Pathology. In FIH, there has generally been involvement of multiple parathyroid glands whether or not synchronous. Parathyroid tumors include polyclonal features, monoclonal features, cystic features in few, and even rare parathyroid malignancy (70). This heterogeneity undoubtedly reflects the unrecognized inclusion of several distinct etiologies within FIH. c. Genes and loci. No gene unique for nonsyndromic isolated hyperparathyroidism has been identified or even mapped. Most families have been too small for genomewide linkage analyses. Virtually each of the few large kindreds with FIH has had a recognized germline mutation in the MEN1, CASR, orhrpt2 gene (70, 72, 76). d. Molecules. Menin, the cas-r, and parafibromin are discussed with their full syndromes (see Sections IV.B.1, IV.C.1, IV.E.1, and IV.E.2). 2. Isolated pituitary tumor a. Expressions. Isolated pituitary tumor has been found in about 100 families (77, 78). Only about five such families are as large as four to nine affected plus unaffected carriers (77, 79). The pituitary tumors are generally GH-secreting. A small fraction of cases have prolactinoma. Because sporadic GHsecreting tumors can cosecrete prolactin, and because prolactinomas seem randomly distributed in the families with GH-secreting tumor, those families with variable fraction of prolactinoma are currently classified as part of this single entity. b. Pathology. The histopathology of familial pituitary tumor is benign and indistinguishable from that of sporadic tumor. 11q13 LOH has been seen in the majority of tested tumors (77) supporting mono- or oligoclonality. c. Genes and loci. A causative gene has not been identified for any family with isolated pituitary tumor. Germline sequencing of MEN1 or PRKAR1A has been done in an index case from many families without finding any mutation (77, 78, 80, 81). Genetic linkage analysis has been reported in only two large families and has been suggested in each gene at 11q13 (thus, near or even identical to the MEN1 gene) (77, 79). LOH testing in the familial tumors has also been compatible with germline or somatic mutation in a tumor suppressor gene at 11q13 (77). 3. Isolated paraganglioma or pheochromocytoma a. Expressions. Paraganglioma is a vascular, usually benign tumor, arising from extraadrenal tissue and associated with the parasympathetic nervous system (82, 83). Paraganglioma is distinguished from pheochromocytoma by its location and its chemical properties. Paraganglioma is typically in the head or neck about the carotid body, the glomus bodies of the jugular bulb, the tympanic plexus, or the vagus nerve. The clinical features depend on the site of origin. Most symptoms of paraganglioma relate to cranial palsies and other mass effects. Uncommonly (about 1%), symptoms arise from systemic catecholamine excess. Familial transmission is recognizable in 10% of paraganglioma cases, and cause by a germline mutation is even more common. Most of the tumors are

7 Marx and Simonds Hereditary Tumors Endocrine Reviews, August 2005, 26(5): paraganglioma, but pheochromocytoma is possible in these families with a varying frequency that depends on the mutated gene. Pheochromocytoma occurs in the adrenal medulla and paraadrenal regions, particularly in the organs of Zuckerkandel. Familial isolated pheochromocytoma (FIPh) has at least nine potential causes: MEN2A, VHL, isolated paraganglioma/pheochromocytoma syndromes from mutation in any of four genes (PGL1 PGL3 are three similar syndromes; and SDHB), NF1, MEN1, and Carney triad. However, among these nine, only VHL type IIC is claimed to be a robust syndrome of isolated pheochromocytoma. The first six have been recognized as occasional causes of FIPh. NF1 and MEN1 should present rarely if ever as FIPh, because other features of each are much more highly penetrant. Incomplete expression of Carney triad as FIPh cannot currently be recognized in the absence of even a mapped locus for that syndrome (see Section IV.B.8). Hereditary pheochromocytoma is bilateral by adulthood in 30 50% of cases but only in those syndromes where the penetrance of pheochromocytoma is high. b. Pathology. The microscopic appearance of paraganglioma or pheochromocytoma is similar. Both are vascular, perhaps due in part to the vascular endothelial growth factor (VEGF) that they overexpress (84). Some 5 10% of paragangliomas or pheochromocytomas become malignant. Malignant behavior correlates poorly with histological appearance. c. Genes and loci. Two familial paraganglioma variants (PGL1 caused by SDHD at 11q22.3, and PGL3 caused by SDHC at 1q21) are transmitted directly from the father, with generation skipping when derived from the mother. This represents an unusual silencing/imprinting mechanism, because the same unaffected and silenced maternal allele seems to be inactivated somatically during tumorigenesis. A third variant (PGL with unassigned number and caused by SDHB at 1p36) differs insofar as it may be only transmitted maternally and has mainly pheochromocytoma. One family classified as PGL2 maps to 11q13, and its mutated gene has not been identified. Germline mutations were searched among 271 cases with apparently sporadic pheochromocytoma. There was germline mutation in 24% overall, with germline mutation of VHL in 11%, RET in 4%, SDHD in 4%, and SDHB in 4%; SDHC was not tested (40). Somatic mutation of RET, VHL, SDHB, or SDHD is only 1 10% each in sporadic pheochromocytoma (85). The mutation patterns in SDHD, SDHC, orsdhb predict loss of function of the encoded protein. LOH at chromosome 11 is usually evident in FIPh tumors and in some 10% of sporadic paragangliomas. This correlates with the locus of SDHD, the main contributing tumor suppressor gene; MEN1 and the gene for PGL2 are also on chromosome 11 but are not known to contribute to a large fraction of these tumors. d. Molecules. Succinate dehydrogenase, also known as succino-ubiquinone oxidoreductase, is the mitochondrial complex II enzyme with four subunits; a separate nuclear gene encodes each subunit. The paraganglioma syndrome has been attributed to an inactivating mutation in three of the four succinate dehydrogenase (sdh) subunits: sdhd, sdhc, or sdhb. 4. Isolated nonmedullary cancer of the thyroid a. Expressions. Sporadic nonmedullary cancer of the thyroid is common. Very few large families that suggest autosomal dominant transmission have been reported (39). Its occurrence in only a few members of a family is thus probably not related to a monogenic cause. Hereditary thyroid cancer at low penetrance can also be an expression from germline mutation in PTEN, APC, WRN, orprkar1a (Table 1), but none of these mutated genes is likely to present as isolated thyroidal cancer; each has other expressions that are far more penetrant. b. Pathology. Like sporadic tumors, hereditary nonmedullary thyroid tumors are mainly papillary. Principally oxyphilic thyroid tumors were noted in one of the two families with linkage to 19p13.2. The hereditary thyroid tumors with polyposis of the colon show excess of cribriform pattern, whereas those in Cowden syndrome or Werner syndrome show excess of follicular pattern (see Sections IV.B.2 and IV.B.4). c. Genes and loci. No mutant gene has so far been identified. Three chromosomal loci for candidate genes for isolated thyroid cancer have been identified, each from genetic linkage testing in one or two large families: NMTC at 2q21, TCO at 19p13.2 [thyroid tumors with oxyphilia (86)], and PTC at 1q21 [with lower penetrance of papillary renal neoplasia (39)]. The locus at 2q21 was delineated in a large Tasmanian family and then supported in 17 of 80 smaller families (87). Dominant multinodular goiter has been seen in two large families with mapping to 14q and Xp22 (39). 5. Isolated MTC a. Expressions. Familial isolated MTC (FMTC), by definition, lacks other clinical features of MEN2A or MEN2B (50). Furthermore, cases with FMTC have MTCs that begin at later age and with lower morbidity than MTC with MEN2A or MEN2B. The differences in expression from MEN2A are particularly striking when only the larger families with FMTC are considered (54, 88 90). Ten percent of cases with FMTC also expressed papillary thyroid cancer, which is high even considering the high rate of discovery incidental to surgery for MTC (91). FMTC can be classified through its phenotype or through its germline mutated RET gene (92). In either case, it can be considered as a variant of MEN2A although it is not literally a MEN syndrome. In several reports, the criteria for FMTC are not stated, and the inclusion of small kindreds increases the likelihood of some families with incomplete MEN2A. In order not to omit the different management appropriate for MEN2A, a definition has been recommended, requiring any FMTC kindred to have over 10 affected members (93). Still the separation of FMTC from MEN2A seems imperfect. For example, one family with RET Val804Leu mutation has met the rigorous size criterion for FMTC (54), but two pheochromocytomas have been reported in another family with the same RET mutation (94).

8 622 Endocrine Reviews, August 2005, 26(5): Marx and Simonds Hereditary Tumors b. Pathology. The pathology of the stages of C cell cancer in FMTC is indistinguishable from that in MEN2A or MEN2B. c. Genes and loci. Isolated MTC has been reported in about 50 families. All but a few families have an identified germline RET mutation (see Section IV.C.2.c). Mutation in the following RET codons has been almost fully specific for FMTC: 630, 768, 790, 791, 804 (Fig. 1) (50). Some of these same RET codon germline mutations have been found in sporadic MTC or sporadic pheochromocytoma (50). In one unusual family, Val804Met RET mutation was not associated with MTC in heterozygotes but was associated with MTC of variable aggressiveness in 3 homozygous carriers (95). d. Molecules. Unlike for the RET mutations in MEN2A and MEN2B (see Section IV.C.2.d), no unique molecular function or molecular domain has been assigned to these RET mutations, possibly specific for FMTC. However, the overall transforming potency in vitro has been lower with mutations associated with FMTC than with mutations causing other MEN2 variants (96). 6. Isolated adrenal-cortical tumor a. Expressions. Autosomal dominant isolated hyperfunction of the adrenal cortex can cause only hypercortisolism or only hyperaldosteronism. Hereditary adrenocortical tumor can also be an expression of MEN1, Carney complex, or Li-Fraumeni syndrome (see Section IV.C.1). FIG. 1.RET gene gain-of-function mutations in few grouped codons cause three variants of MEN2. This figure excludes RET-PTC somatic mutations within the N terminus of RET as a cause of nonhereditary MTC and RET loss-of-function germline mutations anywhere in the RET gene, causing Hirschsprung disease. Representative germline RET mutations are indicated for three MEN2-related phenotypes. b. Pathology. Hereditary isolated tumoral (or type II) hyperaldosteronism in Australia was diagnosed in 67 patients among 27 families, representing a higher prevalence than for nontumoral hyperaldosteronism (97). Because bilateral adrenal hyperplasia has been even more prevalent than unilateral tumor among those cases and because both adrenal features have been diagnosed in the same family, it is unclear whether all these families have one cause and whether the causes are truly monoclonal in the adrenal. Bilateral macronodular disease with hypercortisolism has been reported in one large family (98). Primary (sometimes termed type II) adrenocortical tumor should be distinguished from secondary hyperfunction caused by a steroid biosynthetic defect (sometimes termed type I) with secondary adrenal stimulation by ACTH, angiotensin II, and/or hyperkalemia (36, 99). c. Genes and loci. Genome-wide analysis in 8 members of the largest Australian family with type II or tumoral hyperaldosteronism showed linkage to 7p22 (100). 7. Isolated carcinoid tumor a. Expressions. Isolated carcinoid tumor has not been reported in many members of any family. So the few familial clusters might be random. However, epidemiological analyses have suggested greater than chance association (101, 102). Most of the few reports describe families with two members expressing hindgut carcinoids. In theory, isolated foregut carcinoids (thymus, bronchus, stomach) could be an incomplete expression of MEN1 or NF1. This has not been reported. Two apparently unrelated families each had first degree relatives with bronchial carcinoid but no other features of MEN1 (103). Duodenal somatostatinoma (sometimes termed carcinoid) is rare in NF1 and, therefore, also unlikely to account for several isolated cases in a family. One family has had 3 members, each with multiple duodenal carcinoids (104). b. Pathology. No specific pathological features have been recognized c. Genes and loci. No subchromosomal locus has been mapped for familial isolated carcinoid tumors B. Hereditary hormonal excess in one monoclonal tissue within a multiple neoplasia syndrome 1. HPT-JT a. Expressions. HPT-JT was not recognized until about 1990, and approximately 40 families have been reported. The features are hyperparathyroidism (90%), which includes parathyroid cancer (15%), cemento-ossifying jaw tumors (30%), renal cysts (15%), renal hamartoma [highly penetrant in one family (105)], uterine tumor (455), and Wilms tumor (rare) (70, 76). Progression to chronic renal failure is rare. Among the hereditary syndromes with hyperparathyroidism, HPT-JT stands alone in its high penetrance for parathyroid cancer. This cancer has metastasized systemically as early as age 26 (our unpublished data). Despite their high frequency of malignancy, the parathyroid glands are involved asynchronously; sometimes only a single parathyroid tumor is

9 Marx and Simonds Hereditary Tumors Endocrine Reviews, August 2005, 26(5): found at surgery (70). The parathyroid cancer may be PTH nonsecreting (106). b. Endocrine pathology. About 20% of parathyroid tumors in HPT-JT, whether benign or malignant, show an otherwise unusual micro- or macrocystic component. The renal lesions also are principally cystic, in the form of large cysts that are often bilateral. c. Genes and loci. If a family has been large enough to allow genetic linkage testing in HPT-JT, linkage has always been significant at 1q24-q32, suggesting a monogenic origin (76). The mutated gene in HPT-JT is HRPT2 (76). A small subset of families with apparently isolated hyperparathyroidism has also shown HRPT2 mutation (456). Most sporadic parathyroid cancers have HRPT2 mutation, and an HRPT2 mutation is in the germline in up to half (107). Because the identified germline and somatic mutations all predict premature truncation or absence of the encoded protein, HRPT2 likely contributes to tumors by a loss-of-function mechanism (76). There is 1q LOH in up to half of tested parathyroid or renal hamartoma specimens (105). 1q LOH is also found in 20% of sporadic parathyroid tumors with cystic features, with somatic HRPT2 mutation in 5% of this sporadic and cystic subgroup (76, 108). Immunohistology showed loss of HRPT2 encoded parafibromin in parathyroid cancers (457,458). d. Molecules. The HRPT2 gene predicts a protein (parafibromin) with unknown function. It has a weak amino-acid sequence homology to cdc73p, a yeast protein that is part of a large complex with the polymerase II transcription apparatus (109, 110). A similar complex with parafibromin has been found in human tissues (111). 2. Cowden and Proteus syndromes a. Expressions of Cowden syndrome. Cowden syndrome is a multiple neoplasia/hamartoma syndrome (112). The main features are fibrocystic breast disease (75% of females) with very high risk of malignancy (25 50% of females) and skin lesions (over 90%), particularly trichilemmomas (hamartomas of the hair follicle). Additional features are hamartomatous colonic polyps, early-onset uterine leiomyomata, lipomas, endometrial cancer, macrocephaly, and mental retardation. Non-MTC occurs in up to 10%. Benign thyroid lesions including goiter are more common (50 70%). Thyrotoxicosis is not increased. b. Expressions of Proteus syndrome. The name Proteus is from the Greek god, who could change shape at will. Proteus syndrome includes areas of localized overgrowth, expressed as narrowly as in one digit or as extensively as hemihypertrophy, and with combinations of macrocephaly, facial asymmetry, hyperostosis, and patchy skin changes (11). The skin changes include verrucous nevi, intradermal nevi, hemangiomas, lipomas, and plantar lesions. Ovarian or testicular tumors occur in 25%. Proteus syndrome is included here because of a sometime relation to the PTEN gene (see Section IV.C.2.d). c. Endocrine pathology of Cowden syndrome. The thyroid cancer is usually follicular but can be papillary; this contrasts with the papillary predominance in sporadic and other hereditary non-mtc. d. Genes and loci. Inactivating mutation in the PTEN gene causes all or most cases of Cowden syndrome. Mutations of this gene have also been identified in Bannayan-Riley- Ruvalcaba syndrome (lipomatosis, macrocephaly, and speckled penis) and in two of nine cases of Proteus syndrome (113). The PTEN mutations in Cowden syndrome or Bannayan-Riley-Ruvalcaba are germline; Proteus syndrome is considered a mosaic disorder (see Section IV.C.2.b). Although PTEN mutations are associated with the three described phenotypes, no clear relations to genotype have emerged. Thus, all these patients can be grouped together as PTEN hamartoma-tumor syndrome. LOH at the PTEN locus at 10q22 24 occurs in 25% of sporadic follicular thyroid tumors, but somatic mutation of PTEN there is rare, so any relation to PTEN is uncertain (114). PTEN inactivation contributes clearly to selected sporadic tumors. For example, about half of sporadic endometrial cancers and many colon cancers have somatic PTEN mutation (115, 116). e. Molecule. PTEN encodes pten, a dual specificity lipid phosphatase. In particular it is the 3 -phosphatase for phosphatidylinositol-3,4,5-triphosphate (PI3,4,5P) (117). PI3,4,5P binds to and regulates protein kinase B/Akt and other pleckstrin homology domain-containing regulatory enzymes (118). 3. Adenomatous polyposis of the colon (APC) a. Expressions. APC or familial adenomatous polyposis (FAP) is a syndrome of multiple colonic polyps with high likelihood of progression to cancer in one or more polyp (66). Other features of FAP are desmoids, congenital hypertrophy of retinal pigment epithelium, jaw cysts, sebaceous cysts, and osteomas. Papillary thyroid cancer occurs in approximately 1 2% of cases, or about 10-fold the general population prevalence (39). There is no association with hyperthyroidism. About 90% of thyroid cancer in FAP is in females with average onset at age 25. b. Endocrine pathology. The thyroid tumors in FAP are usually multicentric and papillary, showing in some segments the otherwise unusual cribriform or cribriform-morular variant appearance. Cribriform histopathology is heterogeneous and includes anastomosing arches of cells and solid nests that are unencapsulated, have little stroma, and can infiltrate surrounding parenchyma (119, 120). c. Genes and loci. Germline intragenic mutation of APC is identifiable in up to 80% of index cases with FAP; most of the remainder have large APC deletions that are difficult to detect (66). The APC gene is on chromosome 5q and is inactivated early in colonic tumorigenesis, with biallelic inactivation being identifiable in 80% of benign or malignant tumors in FAP or similarly in sporadic colonic tumors (66). Biallelic

10 624 Endocrine Reviews, August 2005, 26(5): Marx and Simonds Hereditary Tumors inactivation of APC also is found in the associated thyroid tumors. There are modest genotype/phenotype correlations. Most of the thyroid cancers arise in cases with germline mutation in the largest APC exon 15, similar to the genotype of FAP cases with congenital hypertrophy of retinal pigment epithelium (121). Other phenotypes also have distinct genotypes; cases with other extracolonic features have mutation at codons , and those with attenuated FAP have truncations before codon 157. d. Molecules. Apc is a cytoplasmic protein; it binds in vitro to at least seven categories of proteins, including -catenin; the latter seems central. Sporadic papillary thyroid cancer without APC mutation sometimes shows mutation in other genes of the wnt signaling pathway; in particular, it may show mutation of and/or abnormal compartmentalization of -catenin (122). This is particularly true also for the cribriform variant with or without APC (123). Similarly, colon cancers without APC mutation generally have -catenin mutation, suggesting that both genes function in the same pathway, and that disruption in either one is sufficient for an equivalent contribution to this tumorigenesis (124). -Catenin accumulates particularly in the nucleus with APC loss of function. 4. Werner syndrome a. Expressions. Werner syndrome 4 is progeria or accelerated aging with impaired growth (90%), high-pitched voice (80%), early cataract (95%), skin atrophy (90%), soft tissue calcification, osteoporosis (40%), atherosclerosis, diabetes mellitus (50%), graying and loss of hair (30%), and hypogonadism (60%). Werner syndrome is a recessive disorder with greatly increased risk of selected nonepithelial neoplasias (125). They include soft tissue sarcoma, meningioma, melanoma, and osteosarcoma. These neoplasias are the leading cause of death. Thyroid cancer prevalence (10%) is increased, but thyrotoxicosis is not (126). b. Endocrine pathology. Among 23 cases of Werner syndrome with thyroid cancer, only 35% were papillary (127), representing a 3-fold excess of follicular histology, compared with sporadic cases. Cells from Werner syndrome have increased genome instability, seen as chromosomal deletions, reciprocal translocations, and inversions (128). No specific DNA repair system has been implicated. c. Genes and loci. All or most cases of Werner syndrome arise from homozygous mutation of the WRN gene at 8p21. All mutations predict loss of function, and there is no correlation of genotype with phenotype. Mutation of WRN has not been reported in somatic tissues. d. Molecules. WRNp sequence predicts a member of the DExH box family of DNA and RNA helicases. The protein or nucleotide interactions of WRNp are not known. 4 Werner syndrome refers to progeria, sarcomas, etc. Wermer syndrome is MEN1. 5. Li-Fraumeni (cancer family) syndrome a. Expressions. The Li-Fraumeni syndrome is phenotypically heterogeneous (129). Important features include bone or soft tissue sarcoma diagnosed under age 45, breast cancer, brain cancer, and leukemias. Adrenocortical carcinoma occurs in about 1% and usually before age 14. This has been studied in a large group of children with adrenocortical tumor in Brazil. Four Brazilian families each had two to five cases with adrenocortical tumors. Average age at diagnosis of adrenal tumor was 3 yr, and most adrenal tumors were steroid hormone-secreting. About half of the children were virilized, including one fourth with hypercortisolism as well (130). b. Endocrine pathology. Most (72%) of the adrenocortical tumors in the Brazilian familial setting have been malignant (131). c. Genes and loci. The P53 gene is on chromosome 17q. Most kindreds with typical Li-Fraumeni syndrome have a recognizable inactivating germline mutation in the P53 gene. In the large Brazilian cluster of children with adrenocortical tumor, 35 of 36 had the same Arg337His P53 mutation, reflecting common ancestry (132). This mutation has not been reported in other families; thus, it seems to correlate with high penetrance for the adrenocortical tumor phenotype (133). Somatic mutation of P53 is also found in many tumors, including 70% of sporadic adrenocortical cancers (134). A similar cancer family phenotype ( Li-Fraumeni like ) with even greater excess of breast cancer occurs with heterozygous mutation of CHK2/CHEK2. The latter mutated gene can also predispose to familial isolated breast cancer or familial isolated prostate cancer (135). It has not been associated with adrenocortical tumor. d. Molecules. The P53 gene encodes a 393-amino acid nuclear phosphoprotein (66, 129). It belongs to a gene family with P40, P51, P63, and P73. p53 is believed to be a transcription factor that binds to a small DNA motif in the promoter of many genes. 6. Beckwith-Wiedemann syndrome a. Expressions. Cases have macroglossia, abdominal wall defects (including omphalocele), GH-independent macrosomia, and craniofacial dysmorphism. There is increased risk of Wilms tumor, hepatoblastoma, and rhabdomyosarcoma. Neonatal hypoglycemia occurs in 30 60% and usually resolves over 3 d (136). Adrenocortical carcinoma is occasional and can be associated with virilization. One case had bilateral pheochromocytoma (137). b. Endocrine pathology. There are adrenocortical cytomegaly and cysts. IGF-II is overexpressed in several related sporadic neoplasms including adrenocortical tumors and Wilms tumor (36). Neonatal hypoglycemia seems secondary to hyperinsulinism, but the cause of the hypoglycemia needs further clinical analysis (136). c. Genes and loci. The genetic and molecular bases have not been established. Occurrence is sporadic or autosomal dominant with variable penetrance. Mapping in families estab-

11 Marx and Simonds Hereditary Tumors Endocrine Reviews, August 2005, 26(5): lished a locus at 11p15.5 (distinct from WT1 at 11p13), which includes the IGF-II gene. Some cases show uniparental disomy across a large region of 10 Mb, which also includes the IGF-II gene. This particular background occurs in mosaics (138). This is a complex locus that also includes the H19 and p57/kip2 genes. These three and several other genes in this locus are imprinted. Loss of maternally expressed suppressor(s) (such as p57 and/or H19), gain of paternally expressed growth promoter(s) (such as IGF-II), or even combination of the above might contribute to tumor development. d. Molecules. The causative genes and thus their encoded molecules remain unproved. 7. TSC a. Expressions. Principal features of TSC are tuber of brain cortex (93%), subependymal nodule (95%), subependymal giant cell astrocytoma (6%), cardiac rhabdomyoma (50%), renal cysts (10%), renal angiomyolipoma (50%), facial angiofibromas (85%) (formerly misnamed adenoma sebaceum), hypomelanotic macules (more than three in 80%), forehead plaques (25%), nontraumatic ungual and periungual fibroma (20%), and learning disability (50%) (11, 139, 140). Pancreatic islet tumors or cysts are an uncommon but definite hormonal feature. They rarely oversecrete insulin. The Eker rat with a similar syndrome due to a TSC2 mutation expresses nonfunctioning pituitary tumors (141). TSC is included herein because of its occasional hormone-producing tumors and because of its overlaps (mainly nonhormonal) with other hormonal excess syndromes, particularly MEN1 (Table 2A). b. Endocrine pathology. The occasional pancreatic islet cysts and tumors are usually benign. They immunostain mainly for insulin (142). Islet tumor in one case was malignant and invasive when diagnosed at age 12; although apparently hormone nonsecreting, it immunostained for gastrin and, to a lesser extent, for glucagon (143). This latter tumor was associated with a germline mutation in TSC2 and with tumor LOH about the TSC2 locus, supporting its development through biallelic inactivation of TSC2. TABLE 2. Similarities between paired endocrine tumor syndromes c. Genes and loci. TSC can arise from germline mutation in one of two genes, TSC1 on chromosome 9 or TSC2 on chromosome 16. About two thirds of cases arise as new mutations. The disease phenotype from germline mutation in either gene is indistinguishable. Most mutations of TSC1 or TSC2 predict truncation or absence of the encoded protein. Separated by only 6 bp at the 5 end of TSC2 is the otherwise unrelated PKD1 gene, cause of polycystic kidney disease. Contiguous gene syndromes can inactivate both TSC2 and PKD1, causing TSC with renal cysts and often renal compromise (144). In general, biallelic loss of function at the locus for either TSC1 or TSC2 has been demonstrated in many of the various nonhormonal tumors of cases with TSC1 or TSC2 mutation and in some similar sporadic tumors. d. Molecules. TSC1 encodes hamartin, and TSC2 encodes tuberin. Hamartin and tuberin are cytoplasmic proteins that are found as a tight complex with each other. Most diseaseassociated missense mutations of either disrupt this complex (145). Tuberin has a region of GTPase activating protein (GAP) homology. The small GTPase Rheb (Ras homolog enriched in brain) is a direct target of tuberin GAP activity (146). Growth factor signaling through PI3 kinase and protein kinase B/Akt inhibits the GAP activity of tuberin/ hamartin, resulting in Rheb activation (147). 8. Paraganglioma-gastric stromal sarcoma/carney triad a. Expressions. Five families that each include two to three affected members with one or both of multifocal extraadrenal functioning paraganglioma and gastric stromal sarcoma have been proposed to have a novel autosomal dominant syndrome (148). These families were identified among a larger number of cases with the Carney triad, of which the third main feature is pulmonary chondroma. Some cases with Carney triad also have nonfunctioning adrenocortical tumor (13%), esophageal leiomyoma (9%), or other tumor less frequently (149). The full Carney triad has not otherwise occurred on a familial basis and is distinguished by a female sex preponderance. It is not clear whether the Carney triad reflects changes in gene(s) different from that/those specu- A. Syndrome overlap with respect to high penetrance of selected features (mainly mesenchymal tumor) in MEN1 and TSC Tissue Tumor in MEN1 Tumor in TSC Dermis Angiofibroma Collagenoma Angiofibroma Dermal plaque/patch Smooth muscle Leiomyoma Lymphangio-leiomyoma Adipose Lipoma Angiomyo-lipoma Pancreatic islet Islet neoplasm Islet neoplasm B. Carney complex and McCune-Albright syndromes show overlap with regard to major features Tissue Tumor in Carney complex Tumor in McCune-Albright syndrome Anterior pituitary GH GH Thyroid Non C cell thyroid Non C cell thyroid Gonads Sertoli/Leydig androgen-secreting Interstitial/Leydig androgen-secreting Adrenal cortex Nodular hypercortisolism (pigmented) Nodular hypercortisolism (non-pigmented) Myxoma In heart, breast, dermis, etc. In muscle (rare) Melanocyte Lentigine Café-au-lait spot Only selected expressions of each syndrome pair are shown to highlight their overlaps.

12 626 Endocrine Reviews, August 2005, 26(5): Marx and Simonds Hereditary Tumors lated in the five familial clusters. Expressions of paraganglioma were similar to those in other paraganglioma syndromes, including occasional pheochromocytoma (see Section IV.A.3.a). There is a propensity for unusual paraganglioma locations such as the aortopulmonary body. b. Endocrine pathology. Pathological features of the paragangliomas are similar to those in other paraganglioma syndromes. c. Genes and loci. No identification or even mapping of a causative gene has been reported C. Multiple endocrine neoplasia (MEN) syndromes By definition, each MEN syndrome includes neoplasm in at least two different potentially hormone-secreting tissues. Each of these syndromes also includes one or more additional neoplasms. Hormonal excess is the most prominent expression for only two of these syndromes, MEN1 and MEN2 (Table 3). TABLE 3. MEN syndromes have inherent nonendocrine neoplasias Syndrome Endocrine neoplasia Nonendocrine neoplasia MEN1 Parathyroid Enteroendocrine Anterior pituitary Foregut carcinoid Adrenal cortex Angiofibroma Collagenoma Lipoma Leiomyoma Meningioma MEN2 variants Carney complex VHL NF1 McCune- Albright syndrome Thyroid C cell Pheochromocytoma Parathyroid Adrenal cortex Pituitary Gonad Non C cell thyroid Pheochromocytoma Islet tumor Pheochromocytoma Duodenal somatostatinoma Gonadal tumor Somatotrophinoma Non C cell thyroid Neuroma (MEN2B) Myxoma of heart Lentigine Schwannoma Renal cortex cancer Hemangioblastoma Neurofibromas Cafe-au-lait spot Fibrous dysplasia Cafe-au-lait spot For the final four of six syndromes, a nonendocrine tumor is the most important expression. Representative tumors are listed. Bold typeface indicates a prominent clinical feature of the syndrome. 1. MEN1 a. Expressions. The most frequent hormonal tumors in adults with MEN1 are parathyroid adenoma (90%), gastrinoma (40%), and prolactinoma (30%). Carriers are at risk for more than 20 different types of tumor, each inherent to MEN1 (31). Additional hormone-producing tumor associations are insulinoma (10%), glucagonoma, VIPoma, somatostatinoma (each rare), GH (5%), ACTH (2%), TSH (rare), thymic carcinoid (2%), bronchial carcinoid (2%), gastric carcinoid (10%), adrenal cortex (25%), and pheochromocytoma (rare). There are also nonfunctional pancreatic islet tumors in 20%, excluding the remaining 80% with subclinical islet tumors. Selected hormonal tumors (enteropancreatic endocrine tumor excepting insulinoma; foregut carcinoid tumor) in MEN1 have a high malignant potential. In fact, among deaths in MEN1, one fourth have been attributable to an MEN1- related cancer (150). Inherent hormone nonproducing tumors are common but less evident, because they do not cause symptoms from hormone excess; these tumors include facial angiofibroma (88%), truncal collagenoma (72%), lipoma (33%), leiomyoma of the gastrointestinal tract and uterus (uncertain frequency) (31, 64), and meningioma (5%) (459). The types of mesenchymal tumor in MEN1 are similar to those in TSC (Table 2A). b. Endocrine pathology. An overriding feature of MEN1 at the macroscopic level is tumor multiplicity (22, 31). At the time of parathyroidectomy in MEN1, typically three or four parathyroid glands hold tumors. Similarly, gastrinomas (duodenal) in MEN1 tend to be small, submucosal, and multiple; these features, the morbidity of duodeno-pancreatic surgeries, and possibly the presence of occult metastases make gastrinomas in MEN1 usually refractory to surgical cure (151, 152). Gastrinoma in MEN1 shows a different tissue distribution than that of normal gastrin cells (in the gastric antrum). Thus, some consider all extragastric gastrinomas to be ectopic and malignant, independent of histological grade. The less common tumors inherent in MEN1 are generally solitary, including insulinoma and pheochromocytoma. Malignancy of enteropancreatic neuroendocrine of foregut carcinoid in MEN1 is frequent. It is vascular like non-men1 cancers in these tissues. Malignancy in the parathyroid of MEN1 is extremely uncommon, despite multiplicity and early age of onset for the parathyroid tumors (73). There is no established precursor stage for an MEN1 tumor. However, a mouse model for MEN1 has shown striking -cell hyperplasia as an insulinoma precursor stage (153). c. Genes and loci. All typical MEN1 cases probably arise from germline mutation in the same gene. The MEN1 gene is at 11q13 and contributes to tumors mainly via a biallelic loss-of-function mechanism. Eighty percent of the germline or somatic MEN1 mutations predict truncation or absence of menin protein; the remaining 20% of germline mutations predict change of one or few codons (missense) and are also likely to cause loss of menin function. No clear genotypephenotype correlation has been recognized; however, among 19 germline MEN1 mutations found in the minority of all families with isolated hyperparathyroidism, there has been a mild clustering of missense mutations about codons (70, 154). Most of the intrinsic tumors in MEN1 have biallelic MEN1 inactivation (31). This is generally suggested by 11q13 LOH. Rarely, the wild-type allele is inactivated by a small MEN1 somatic mutation that does not result in a zone of 11q13 LOH (155). 11q13 LOH has not been found in the adrenocortical tumors or thymic carcinoids that are clearly associated with MEN1 (156, 157), raising the possibility of two specific tissue types with a differing step of tumorigenesis. The adrenocortical tumors in MEN1 are generally benign, whereas the mediastinal thymic carcinoids are generally malignant. Among related sporadic tumors, biallelic somatic mutation of the MEN1 gene is common in parathyroid adenoma (20%), insulinoma (10%), gastrinoma (20%), and bronchial carcinoid (40%); however, it is rare in anterior pituitary tu-

13 Marx and Simonds Hereditary Tumors Endocrine Reviews, August 2005, 26(5): mor (2%), follicular thyroid tumors (below 2%), leiomyoma (64), or small cell lung cancer (31, 158). d. Molecules. The MEN1 gene encodes menin, a widely expressed protein of 613 amino acids. Menin resides mainly in the nucleus and has been localized to telomeres during meiosis (159). The physiological functions of menin are not known, but menin can interact in vitro (directly or indirectly and with unknown importance) with a growing list of more than 10 proteins (160). Menin partners with the strongest suggestions for involvement in tumorigenesis are jund and a large complex, homologous to the COMPASS complex in yeast ( , 460). 2. MEN2A and MEN2B a. Expressions. MEN2A consists of MTC (95%), pheochromocytoma (50%), and hyperparathyroidism (30%). MEN2B has the features of MEN2A, plus intestinal ganglioneuromas and the mucosal neuroma phenotype (90%), but minus hyperparathyroidism (50). Most aspects of FMTC are considered separately (see Section IV.A.4). Each of the three is a variant of MEN2. The relative prevalences of index cases are MEN2A MEN2B FMTC (50, 93, 164). Late dissemination of MTC can be to lungs, liver, and/or brain. The relative ordering of MTC for lower age of onset and greater aggressiveness is highly syndrome-dependent: MEN2B MEN2A FMTC. Earliest age of known dissemination of MTC in a variant of MEN2 has been central in determining the earliest age for suggesting prophylactic total thyroidectomy for carriers. Metastases of MTC have been recognized as early as the age of 1 yr in MEN2B, as early as age 5 yr in MEN2A, and rarely before age 15 yr in FMTC. Pheochromocytoma was formerly a major source of morbidity, similarly in MEN2A and MEN2B. This is no longer true because of prospective monitoring and improved pharmacological managements, particularly around surgery. Pheochromocytoma causes more symptoms in MEN2 than in VHL; this correlates with higher tumor expression of tyrosine hydroxylase and more tissue stores of catecholamines in MEN2 (165). Among the three main hormonal excess states of MEN2A, hyperparathyroidism causes the least morbidity. Parathyroid involvement in MEN2A can reach 50% or more by age 70 yr and can be in multiple glands (166, 167). Parathyroid tumors are more asynchronous in MEN2A than in MEN1. b. Endocrine pathology. C cell neoplasia begins as multifocal hyperplasia and then becomes micronodular. C cell cancer can also be multifocal in MEN2 (168). In fact, it has been suggested that monoclonality within C cell hyperfunction is present at the stage of hyperplasia and might even begin at embryonic stages before lobation of the thyroid (169). Eventual spread is to regional lymph nodes. Pheochromocytoma in MEN2 also begins as a hyperplastic stage before progression to mono- or oligoclonality as evidenced by biallelic inactivation at the VHL locus and by LOH at 1p (170); a similar implication of additional loci has not been seen with pheochromocytoma in VHL (171). This tumor often remains histologically benign but sometimes aggressive metabolically. Monoclonality of the parathyroid tumor in MEN2A has not been documented. c. Genes and loci. In the recent past, calcitonin assay after challenge with pentagastrin was used as a sensitive and semiquantitative index for MTC, C cell hyperplasia, and thus for ascertainment of the MEN2 carrier state. MEN2 carrier ascertainment by serum calcitonin protocols has been largely replaced by the more effective sequencing for germline RET mutation (50, 93). The RET gene is at chromosome 10q11.2, and one of its germline mutations is detectable in 98% of index cases with an MEN2 variant (50, 93). In fact, the RET locus has never been excluded by chromosome 10 haplotyping in any MEN2 family. The Cys634 codon accounts for 80% of germline mutations in MEN2A, whereas the Met918 codon accounts for 95% of germline mutations in MEN2B (Fig. 1). RET mutations are found in somatic tissues of sporadic MTC or sporadic pheochromocytoma, but with a very different frequency distribution of mutated RET codons. Met918 is the most frequent somatic RET mutation in sporadic tumors and accounts for 25% of the RET codon mutations in these (50). Somatic gain-of-function RET missense mutation is found in about one fourth of sporadic MTC and one tenth of sporadic pheochromocytomas. It has not been found in sporadic parathyroid adenoma. RET-PTC, a different mechanism of somatic RET gain-offunction mutation, leading to a ret fusion protein, is common in sporadic nonmedullary papillary thyroid cancer (see Section IV.C.2.d). d. Molecules. Ret protein with four tandemly repeated extracellular cadhedrin-like domains is a transmembrane tyrosine kinase, part of a large family that includes the C-MET oncogene. Upstream and downstream components of the normal ret pathway have begun to be clarified (see Section VII.G.2) (18). The ret system is functional during organogenesis in intestinal ganglia and the kidney. This correlates well with loss-of-function mutations in RET that are a major cause for familial Hirschsprung disease (intestinal aganglionosis); it is thus an interesting paradox that occasional cases of MEN2A (with presumed RET gain-of-function) also express Hirschsprung disease (18). There is a striking correlation of RET genotype and MEN2 phenotype (Fig. 1). Most germline RET mutations in MEN2A are at cysteine residues in the cysteine-rich extracellular domain about cysteine-634. Most MEN2B RET germline mutations are at methionine-918 at its cytoplasmic tyrosine kinase catalytic site. RET germline mutation in FMTC occurs in a zone that spans both of those domains but shows an extra location, almost specific to FMTC at codons All MEN2-associated RET mutations predict missense, and all are believed to cause ret gain of function (18). The extracellular mutations probably cause gain of ret function by promoting ret dimerization; the MEN2B-associated mutations in the cytoplasmic tyrosine kinase domain activate the tyrosine kinase directly. Tyrosine kinase inhibitors have already been targeted to certain cancer genes, and the RET gene is under exploration for this (172). Somatic but not germline RET mutation is common in

14 628 Endocrine Reviews, August 2005, 26(5): Marx and Simonds Hereditary Tumors papillary non-mtc. Most sporadic papillary (and some Hürthle or follicular) thyrocyte tumors have a 5 (i.e. aminoterminal) fusion of RET with one of eight other genes. The fusion genes are termed RET-PTC1 through RET-PTC8 (173, 174). Progression to poorly differentiated thyroid cancer is rare with RET-PTC (175). Each of these eight gene fusions can have any among three consequences that favor RET gain of function: 1) a new promoter drives ectopic expression in thyrocytes; 2) abnormal compartmentalization in cytoplasm is favored by loss of the transmembrane domain; and 3) the fusion protein has a new N terminus, favoring homodimerization. 3. VHL a. Expressions. VHL is a highly penetrant disorder with apparent age of onset that varies with the family and the thoroughness of evaluations (11, 176). The main features of VHL are nonhormonal (Table 3): clear cell (sometimes with occasional granular cell) renal cancer (40%), renal cysts (35%), cerebellar and spinal hemangioblastoma (60%), papillary cystadenoma of the epididymus or broad ligament (15%), and retinal angioma (60%). Retinal angioma is typically the feature with earliest age of onset during thorough prospective screening. The renal carcinomas are multifocal and often will recur with current management by conservative or kidney-sparing resection. The hormonal features of VHL are pheochromocytoma (20%) and pancreatic islet tumor (10%). Pheochromocytoma clusters only in selected VHL families, leading to their classification as VHL type II; furthermore, those rare families with VHL mutation and with only pheochromocytoma are classified as VHL type IIC (176). Pheochromocytoma in VHL may become symptomatic during childhood. The catecholamine-related biochemical profile differs between pheochromocytomas of VHL and MEN2A (see Section IV.C.2.a). Pancreatic islet tumor in VHL as in TSC does not usually oversecrete hormones but presents as a mass. b. Endocrine pathology. The nontumorous adrenal medulla in VHL, unlike in MEN2, does not show chromaffin hyperplasia, and it shows a characteristic amphophilic or clear cytoplasmic morphology (177). The pancreatic islet tumors are generally benign and multiple. Islet histology is micro- or macrocystic, serous, or adenomatous. Many islet tumors or cysts in VHL immunostain for insulin but rarely, if ever, cause hypoglycemia (178). The hormonal and nonhormonal tumors of VHL have either large vessels or a prominent vascular component; this seems related to their overexpression of VEGF (179). c. Genes and loci. A VHL germline mutation is identifiable in virtually all VHL families. All of the germline and tumorassociated somatic VHL mutations likely cause VHL loss of function. Some cases without identified mutation are mosaics (180). Over 95% of VHL families with pheochromocytoma (type II) have VHL missense mutations that cluster in a small part of the 3 end of the open reading frame. VHL is a typical tumor suppressor gene. LOH that includes the germline locus (at chromosome 3p) is generally identifiable in the monoclonal neoplasm as well as in the cyst of VHL. However, unlike most other tumor suppressor genes, the VHL gene often shows somatic inactivation without LOH but instead shows VHL gene hypermethylation; this occurs in familial or sporadic tumors (181). A very different phenotype, accounting for most cases of congenital polycythemia, results from germline biallelic missense VHL mutation, usually including one or both copies of 598C3T (182). These cases have not shown features of VHL. d. Molecules. pvhl is part of a multisubunit complex with E3 ubiquitin ligase activity; SCF complexes include Cul1 and Skp1, whereas alternate complexes (VCB-Cul2) include Elongin B, Elongin C, and Cul2. A major substrate of the pvhl-associated ubiquitination is hypoxia-inducing factor (HIF)-1 or HIF-2. Direct binding with HIF- is in a pvhl pocket that is also a hotspot of pvhl mutations (183). HIF- s are transcription factors that mediate the hypoxia response, including expression of VEGF (see Section VII.G.3). In the absence of pvhl activity, HIF- s accumulate because their degradation is impaired. VHL mutations associated with the VHL phenotype of pheochromocytoma only (VHL type IIC) conserve major aspects of the interaction of pvhl with HIF- (see Section VII.G.3), suggesting that other interactions might be disrupted by these mutations (184, 185). 4. Carney complex a. Expressions. Two thirds of cases with Carney complex have cardiac myxoma. These myxomas can be multicentric and can recur after resection (186). Cardiac myxomas can be lethal and account for half of sporadic cardiac neoplasms. Some 7% or more of cardiac myxomas are in the hereditary setting of Carney complex. Cutaneous myxomas are also common, particularly about the eyes, head, and breasts (33%). Many (75%) cases express punctuate melanotic skin pigmentation, particularly on the vermillion borders of lips and center of the face. Psammomatous melanotic schwannoma occurs in 50% but is otherwise rare. Hormonal hyperfunction in Carney complex is somewhat less common than nonhormonal hyperfunction (187) (Table 3). Cushing s syndrome due to bilateral primary pigmented nodular adrenocortical disease (25%) is virtually pathognomonic. Approximately 10% have GH-secreting pituitary tumor with rare oversecretion of prolactin. Approximately 10% have thyroid hyperfunction that is usually silent clinically in the form of benign neoplasms, cysts, and rarely cancer. Sertoli tumors (mainly the large-cell calcifying variant) of the testis occur in 33% of males. Leydig cell hyperplasia is uncommon (C. Stratakis, personal communication). Cystic ovarian lesions without steroid oversecretion occur in 60% of women. The many features of Carney complex result in diverse composite expressions but high overall penetrance in an autosomal dominant pattern with expression typically by age 20. The hormonal and nonhormonal features overlap with those of McCune-Albright syndrome (Table 2B); the molecular defects in both syndromes impact on the camp/ protein kinase-a (PK-A) signaling system (see Section VII.G.2). b. Endocrine pathology. Pigmentation in lesions of the dermis, but not adrenal, reflects increased melanin content; these lesions are usually benign. Adrenal weight varies from decreased to mildly increased, with countless small, pigmented

15 Marx and Simonds Hereditary Tumors Endocrine Reviews, August 2005, 26(5): cortical nodules (below 4 mm). The adrenal nodules are composed mainly of enlarged cortical cells with granular cytoplasm that often contains lipofuscin. The internodular cortex is atrophied and disorganized (188). c. Genes and loci. Genetic linkage analysis in Carney complex identified two loci of candidate genes, 2p16 and 17q24. Additional genetic heterogeneity is evidenced by several families, whose linkage analysis excluded either locus (186, 189). The mutant gene at 17q24 is PRKAR1A, which encodes the previously well-characterized R1- regulatory subunit of PK-A (190). Germline mutations in PRKAR1A account for half of index cases with Carney complex. Only subtle relations of genotype and phenotype have been recognized (191). Identified PRKAR1A mutations in Carney complex all predict truncation or absence of the protein; no missense mutations have been identified. These mutation types and the finding of 17q LOH in syndromal tumors each suggest that PRKAR1A contributes to tumor through loss of function. Several chromosomal regions can display clonal rearrangements in tumors of Carney complex. These include amplification in a region of 2p that overlaps the locus for one unidentified Carney complex candidate gene (192); this is suggestive of gain of function of an oncogene at this locus. LOH at 17q24 has been identified in less than half of syndromal tumors, including in about half of those with germline mutation at that locus (193). Somatic mutation of PRKAR1A (see Table 1) is rare in sporadic tumor of the pituitary or thyroid (80). d. Molecules. PRKAR1A encodes one of four homologous regulatory subunits for the PK-A enzyme (camp-dependent protein kinase). The subunits have a domain that can be a substrate for the protein kinase-a enzyme and thereby can bind to and occlude the active site of the catalytic subunit. This inhibition is terminated after a regulatory subunit binds camp and becomes dissociated from the catalytic subunit. 5. NF1 a. Expressions. NF1, also known as von Recklinghausen NF or peripheral NF, is a common autosomal dominant disorder (prevalence, 1 in 3000) with near 100% penetrance and variable expressivity (11, 194). The commonest features are caféau-lait spots (six or more, greater than 1.5 cm, postpubertally, and mostly with smooth borders in 95%), peripheral neurofibromas (95%), freckling of axilla or groin (67%), Lisch nodules (tan benign tumors) of the iris (90%), plexiform neurofibroma arising from deep nerve trunks (30%), learning difficulty (30%), and bone lesions (5%). Less common features include hormonal tumors. Pheochromocytoma occurs in a small fraction (1 2%). Duodenal carcinoid or somatostatinoma of the ampulla of Vater is also uncommon (below 1%) (195); in fact, about one third of the latter, otherwise rare tumors, are in NF1. Primary hyperparathyroidism also is unusual in NF1 (below 1%) (196). None of these three hormonal tumors has been reported in a case with NF2 (also termed central NF). Precocious puberty or GH excess can rarely occur in NF1 secondary to optic glioma with diencephalic dysfunction. b. Endocrine pathology. Pheochromocytoma has been proved to be an inherent feature of NF1 by its modest frequency therein and by 17q LOH, suggesting biallelic NF1 loss of function (36). The critical cell for tumorigenesis of pheochromocytoma in NF1 may be the supportive Schwann cell, because neurofibromin is absent therein but overexpressed, surprisingly, in the chromaffin cells of the tumor (197). This is supported by Schwann cell neurofibromas in mice with selective knockout of neurofibromin from the Schwann cell (198). Similar evaluations of DNA or other molecules have not been reported in NF1 for other possibly associated endocrine monoclonal neoplasms: duodenal somatostatinoma or parathyroid adenoma. c. Genes and loci. The NF1 gene has at least 57 exons and an open reading frame of 9 kb. This large size has compromised efforts at mutation analysis. Approximately half of index cases represent new mutation, with the majority arising in the paternal allele. No correlation of phenotype and genotype has been recognized. Most of the recognized mutations predict neurofibromin protein truncation or absence. NF1 mutation has not been established in sporadic tumor, but this needs further evaluation, particularly in juvenile myelomonocytic leukemia (199). Although 17q LOH in these settings may support a biallelic loss-of-function mechanism, the nearness of NF1 to the P53 gene on chromosome 17p renders this inconclusive. d. Molecules. The predicted protein from NF1, termed neurofibromin, has 2818 amino acids. Neurofibromin is widely distributed with a concentration in neural tissues, particularly astrocytes (200). It colocalizes with microtubules, a cytoplasmic component. It has GTPase activator homology and activity. Such activity toward ras or another GTPase could account for its tumor suppressor properties. It may also have a close interaction with merlin, the product of the NF2 gene (201). D. Mosaic states One explanation for variable or even undetectable expression of a disorder in a carrier is mosaicism. Mosaicism implies that the subject is a mixture of two or more cell lines that derived from one fertilized egg. By definition, mosaicism cannot exist in a gamete because this is one cell; thus, mosaicism cannot be transmitted through the germline. Mosaicism arises by postzygotic mutation in contrast to chimerism that is a mixture of cell lines from two or more fertilized eggs, as after a bone marrow allotransplant. Mosaicism is likely in some multiple neoplasia disorders with variable expression and lack of transmission in some families. This includes Beckwith-Wiedemann syndrome (see Section IV.B.6), Proteus syndrome (see Section IV.B.2.b), VHL (see Section IV.C.3), segmental NF1 (see Section IV.C.5), and Sturge-Weber syndrome (not discussed further). In particular, the McCune- Albright syndrome seems always to be a mosaic condition. 1. McCune-Albright syndrome a. Expressions. The two most frequent features of McCune- Albright syndrome are nonhormonal: polyostotic fibrous dysplasia of bone and multiple café-au-lait spots (202) (Table

16 630 Endocrine Reviews, August 2005, 26(5): Marx and Simonds Hereditary Tumors 3). Fibrous dysplasia of bone presents in childhood with pain and fracture. The café-au-lait spots rarely cross the midline and follow a characteristic segmental pattern that correlates with the developmental lines of Blaschko (202). The full triad includes precocious puberty [in female more than male (M. Collins, personal communication)] secondary to primary gonadal oversecretion of sex steroids. Selected components of the syndrome, such as monoostotic fibrous dysplasia of bone, are more common than the full triad and often have the same mosaic pathophysiology. Cases with two of the three principal features are usually considered to have the syndrome. An uncommon feature that strengthens the tissue overlap with Carney complex is im myxoma (203) (Table 2B). Precocious puberty is more common than its other hormonal expressions, including hyperthyroidism often with a nodular thyroid, ACTH-independent hypercortisolism, GH- or prolactin-oversecretion, and tumor-associated hypophosphatemia. Hypercortisolism is uncommon and presents before the age of 1 yr. The hypophosphatemia is presumed to result from phosphatonin release by bone lesions; conceptually, this FGF23 excess can be considered like hormonal excess associated with a monoclonal tumor (204). The above expressions imply that McCune-Albright syndrome is a congenital MEN syndrome, albeit not a hereditary one. b. Endocrine pathology. Endocrine pathology of the McCune-Albright syndrome has not been studied in detail. Nodular hyperplasia of the thyroid has been described (205, 206). The ovaries have large follicular cysts without ovulation (202, 207). The stimulatory isoform of the G protein (Gs) normally is expressed only from the maternal allele in the pituitary (208) and proximal tubule of the kidney (209). Studies with fibroblasts from fibrous dysplasia have suggested that the mutant skeletal fibroblast can only survive after transplantation in vivo as an admixture with normal fibroblasts (210). This suggests that some or all McCune-Albright tumors develop differently from other monoclonal tumors. The mosaic origin, of course, represents a mutant clone contributing to each lesion. In contrast, the occasional progression to sarcoma is an extreme expression of monoclonal growth, perhaps reflecting additional somatic mutations and perhaps independent of admixed normal cells. c. Genes and loci. McCune-Albright syndrome and its incomplete forms, such as monoostotic fibrous dysplasia, arise from heterozygous gain-of-function mutation in GNAS, which encodes Gs, the -subunit of the stimulatory G- protein heterotrimer. d. Molecules. Mutation of codon 201 in Gs slows the hydrolysis of bound GTP and thereby prolongs the active state of Gs. Gs with gain-of-function mutation is likely to signal in excess to any of its several effector molecules. These can include adenylyl cyclase with subsequent camp-mediated activation of PK-A or camp activation of Epac1 and Epac2 (exchange factors for the Rap small GT- Pase) (211, 212), the Src and Hck nonreceptor tyrosine kinases (213), and possibly the Gs -specific RGS protein RGS-PX1 (214). Although substitutions at both arginine- 201 and glutamine-227 can activate Gs, only mutation at arginine-201 has been seen in McCune-Albright syndrome. Identical mutations in GNAS have been common in isolated fibrous dysplasia of bone as well as in sporadic GH-secreting pituitary tumor. As a widespread signal transduction molecule, Gs has effects that are not restricted to hormone secretory cells. 2. Segmental NF1. Some 150 cases have been reported with NF1 limited to one or several body segments (11). Usually the specific feature of segmental NF is cutaneous. E. Hereditary syndromes with polyclonal hyperfunction of one hormonal tissue 1. FHH a. Expressions. FHH causes lifelong hypercalcemia (100%), normal serum PTH despite hypercalcemia (90%), normal urine calcium despite hypercalcemia (i.e., relative hypocalciuria) (90%), mild hypermagnesemia (30%), and persistent hypercalcemia after subtotal parathyroidectomy (100%) ( ). Uncommon expressions are high PTH (10%), serum calcium above 14 mg% (below 5% of cases), recurrent pancreatitis (less than 1%), and neonatal severe primary hyperparathyroidism (NSHPT; rare) (see Section IV.E.2). When the parathyroid cell of FHH is challenged with a range of extracellular calcium in vivo or in vitro, the PTH secretory response shows a shift of the calcium set-point to higher calcium. A tenacious PTH-secretory regulation by even a markedly reduced parathyroid cell mass explains the persistence of hypercalcemia after subtotal parathyroidectomy. FHH is a form of primary hyperparathyroidism, because hypercalcemia is caused by inappropriate PTH (216). However, the hyperparathyroidism is atypical due to most of the above features. b. Endocrine pathology. Typically, the parathyroid glands all appear normal. Between 10 and 20% of the parathyroid glands are mildly enlarged. Even the normal size and histology represent parathyroid function that is fundamentally excessive, considering the lifelong hypercalcemia (218, 219). Parathyroid lipohyperplasia is seen occasionally. Parathyroid gland nodularity has not been seen, and asymmetric enlargement is rare. Although clonality in the typical parathyroid gland of FHH has not been tested, a normal size and uniform histology and other expressions mentioned suggest that the parathyroid gland hyperfunction is polyclonal. Immunohistology of the cas-r expression has not been done in FHH. One large and unusual family with CASR missense mutation has shown features of more typical hyperparathyroidism with nephrolithiasis and monoclonal parathyroid neoplasia (74, 75). c. Genes and loci. FHH is usually caused by mutation in the CASR gene on 3q22 (45, 215, 220). The mutations sequence and their expression in vitro predict loss of function of the CASR, i.e., function as a tumor suppressor; surprisingly, mutation of the CASR occurs rarely if ever in sporadic para-

17 Marx and Simonds Hereditary Tumors Endocrine Reviews, August 2005, 26(5): thyroid adenoma (221). About one third of FHH kindreds linked to 3q22 do not have an identifiable CASR mutation, suggesting other types of mutation in the CASR. In contrast, genetic linkage in two large families with FHH has pointed to two other causative genes at 19p and 19q (222, 223). d. Molecules. The CASR encodes the cas-r, expressed widely but most densely on the parathyroid cell plasma membrane and less so on the renal tubules. It mediates calcium-sensing in the parathyroid and the renal tubule, and its germline mutation accounts for the expressions of FHH in those tissues. Agonists at the cas-r (calcimimetic drugs) are under active development as possible treatments for primary and secondary hyperparathyroidism (224, 225). The cas-r can act directly in vitro on either of two GTP-binding transducers, Gq and Gi (226). However, its partners in vivo and the remainder of its downstream pathway in the parathyroid cell have not been identified. The unidentified FHH loci at chromosome 19p and 19q could encode two proteins, one or both of which is specific for the same pathway. Three families had FHH with autoantibodies against the cas-r and with other immune features (Hashimoto thyroiditis and celiac sprue). No affected member of these three families had mutation of the CASR (227, 228). 2. NSHPT a. Expressions. NSHPT is a life-threatening disorder (157, 215, 217). The criteria for defining it are important in determining the fraction with each of its several etiologies. We define NSHPT as serum calcium concentration in the range of 16 mg% or higher in a neonate with very high PTH. The general expressions are hypotonia (80%), bell-shaped chest and/or rib fractures (70%), respiratory distress (70%), relative hypocalciuria (80%), and family history of FHH, hypercalcemia, or parental consanguinity (80%). Only rare cases have survived without early total parathyroidectomy and then with severe morbidity (229). One group to exclude from NSHPT is FHH neonates with moderate hypercalcemia and variable distress. Most of these are the offspring of unaffected mothers; they likely express the consequences of secondary hyperparathyroidism in response to normal maternal serum calcium in utero, that was perceived as inadequate by the fetus. If supported or left untreated, such cases can recover to show typical asymptomatic FHH. b. Endocrine pathology. The parathyroids are symmetrically and markedly enlarged. In theory, such enlargement could begin in utero. Interestingly, in the mouse model with CASR homozygous knockout, parathyroid size only becomes enlarged after birth (230). The parathyroids show diffuse hyperplasia of chief cells without nodularity. Because parathyroid hyperplasia in other settings can have monoclonal overgrowth, parathyroid monoclonality in these neonates seems remotely likely but was not tested (231). c. Genes and loci. Most cases are homozygous for germline inactivating mutation of the CASR. Similar cases have occasionally been compound heterozygotes for two CASR mutations. The null status of the CASR has been supported by similar expressions after homozygous knockout of the CASR in mice (230). In one apparent CASR / heterozygote, cas-r function as a dominant negative allele was suggested (232). d. Molecules. See Section IV.E.1.d. 3. Nonimmune hyperthyroidism a. Expressions. Also termed hereditary toxic thyroid hyperplasia, this autosomal dominant disorder shows marked variability even within a family (233). The hyperthyroidism begins anytime from infancy to adulthood. The variability within families probably relates to interacting genetic and environmental factors such as iodine intake. There is a diffuse goiter that progresses. The hyperthyroidism does not remit spontaneously; in fact, it recurs after partial treatments. Only aggressive ablation (radioiodine or surgery) followed by thyroid hormone replacement can be curative. b. Pathology. The thyroid shows a diffuse goiter with no lymphocytic infiltration and no nodularity. Progression to malignancy has not been reported. Although clonality has not been tested, the histological appearance suggests a polyclonal process. c. Genes and loci. Nonimmune hyperthyroidism is caused by heterozygous gain-of-function mutation in the TSHR gene in chromosome 14. Occasionally this arises sporadically through new germline mutation; the most severe of these have been congenital presentations and have not been familial. A very similar spectrum of somatic TSHR mutations has been noted in a large fraction of sporadic thyroid toxic adenomas and in a similar fraction of nodules in nodular goiter; in the latter condition, different mutation may be present in independent nodules and even in areas without a discrete capsule (57, 58). Activating mutation of the TSHR is rare in sporadic thyroid cancer, with only occasional contribution to sporadic follicular thyroid cancers (234). d. Molecules. The TSH receptor (TSHR) belongs to the large family of serpentine or G protein-associated transmembrane receptors. Most TSHR-activating mutations are in the third cytoplasmic loop or in the adjacent sixth transmembrane segment. This clustering reflects an important role of these domains in activation of this receptor (233). Features tested in vitro and common to most activating TSHR mutations are: 1) higher binding affinity than the normal TSHR for TSH; 2) decreased EC 50 for TSH responses; 3) an increase in basal adenylyl cyclase activity in vitro; and 4) decreased expression on the thyroidal cell surface. 4. Testotoxicosis a. Expressions. Testotoxicosis or familial male-limited precocious puberty is transmitted in an autosomal dominant male-limited pattern (235, 236). The precocious puberty usually begins before age 4 with high testosterone levels independent of serum gonadotropins, which remain at low prepubertal levels. Normally, LH is sufficient to stimulate androgen production in males, but both FSH and LH are necessary to stimulate ovarian steroidogenesis; this explains why female carriers of a mutant activated LHR do not express precocious puberty. Androgen-directed drugs (synthesis blockers, receptor blockers, or both) are the principal treat-

18 632 Endocrine Reviews, August 2005, 26(5): Marx and Simonds Hereditary Tumors ments. Ablation of the testes is considered too invasive to be used in this setting. In fact, the affected males generally are fertile. b. Pathology. Associated Leydig cell hyperplasia can be diffuse or nodular. Progression to adenoma or cancer has not been noted. c. Genes and loci. Some 15 activating mutations have been found in eight codons of the LHR (235, 236). Activating LHR mutation has also been noted in several sporadic Leydig cell adenomas (237). d. Molecules. Most of the activating mutations of the LH receptor (LH-R) are in the cytoplasmic side of the sixth transmembrane domain; this is similar to the distribution of mutations of the TSHR in nonimmune hyperthyroidism. But the TSHR can be activated at many other codons. Biological expression of mutant LHR is generally measured with stable transfection of LHR constructs into cell lines. Most of the testotoxicosis-associated LHR mutations result in increased basal and agonist-dependent signaling in vitro; this is mainly detected as LH-responsive increases in intracellular camp and sometimes phospholipase C activity. The mutation (Asp578Tyr) that is most severe in vitro causes precocious puberty the earliest (before age 1 yr); a mutation (Met398Thr) with generally mild effects in vitro is otherwise associated with some male carriers that are asymptomatic. Lastly, the LHR mutation (Asp578His) found in sporadic Leydig cell adenoma is the most active in vitro, where it strongly stimulates camp and phospholipase C. 5. PHHI a. Expressions. PHHI is clinically and genetically heterogeneous. The most severe expressions begin in neonates; these have fetal overgrowth and a 25 50% likelihood of consequent neurological deficits and/or seizures (238). An equal proportion has onset later in infancy, and this does not distinguish the various genetic etiologies. Despite onset in infancy, milder features may delay recognition to age 1 2 yr (239). There is a characteristic squared facies in cases with neonatal or infantile onset; similar facies have not been noted in other hyperinsulinemic states with fetal overgrowth (240). A subgroup representing about 5% has 3- to 4-fold increase of serum ammonia (reflecting hepatic expression of GLUD1 mutation (241). The majority benefits little from near total pancreatectomy. In contrast, a clinically indistinguishable subgroup of about 30% has been diagnosed and operated after any of a series of insulin localizing procedures such as selective venous sampling for insulin. Their pancreatic disturbance is local in the pancreas and not diffuse. Focal pancreatic resection after an abnormal localizing test in this subgroup carries a very high cure rate (242). b. Endocrine pathology. The underlying pancreatic pathology of PHHI was formerly referred to as nesidioblastosis or nesidiodysplasia (21), but this is no longer considered specific nor is the -cell proliferation rate even increased, according to Ki-67 labeling index (24). Furthermore, the pathology has not been fully examined alongside the current classification based on one of four mutated genes. The general findings in the 50 80% of cases with diffuse pancreatic islet involvement are enlargement of islet nuclei, cellular crowding, and normal -cell proliferation rate (23). The most important feature is the diffuse nature of the islet disturbance throughout the pancreas. This is probably a polyclonal process. A distinct subgroup of 20 50% of cases have a focal islet lesion that is nodular with increased -cell proliferation, whether or not it is termed focal nesidioblastosis or, more recently, focal nodular islet hyperplasia. This is a mono- or oligoclonal process (24, 243). When onset is even later in childhood, typical insulinoma is characteristic (239). None of the four genes implicated in PHHI has been examined for somatic mutation in insulinoma (Table 1). Nesidioblastosis beyond the fetal stage (where it is normal) is rarely documented (244). An association of nesidioblastosis with pancreatic ductular adenocarcinoma but not hypoglycemia was seen in one family with 17 affected members (245). Nesidioblastosis has also been reported in a small number of adults with hypoglycemia and nontumoral hyperinsulinemia (246); such classification is controversial (247). c. Genes and loci. Mutation in any one of four genes has been implicated in PHHI (248) (Table 1). None of these four has yet been implicated in sporadic insulinoma, but this needs testing in a large series of tumors. The commonest genes to be mutated, both at chromosome 11p15, are SUR1 (ABCC8) and Kir6.2 (KCNJ11) in roughly 50 and 10%, respectively (239). Either may be inactivated by a homozygous or by a compound heterozygous mutation. Most mutations of SUR1 or Kir6.2 predict truncation or absence of its encoded protein, but several milder cases have had missense mutations (249). PHHI can also result from heterozygous gain-of-function mutation of GK or of GLUD1. GK (GCK or MODY2) (on chromosome 7) can be activated rarely (only two reported index cases) in a heterozygous gain-of-function mutation, with variable presentation from infantile hypoglycemia to asymptomatic adult within one family (250). GLUD1 (on chromosome 10q) also can be activated through heterozygous gain-of-function mutation (251). SUR1 (like Kir6.2) is an imprinted gene at 11p15. All or most cases with PHHI and associated focal nodular islet hyperplasia have had germline mutation of the paternal SUR1 allele. The second SUR1 allele is consistently inactivated by allelic loss of the maternal copy (240). This hypoglycemic trait has not been reported in more than one member of a family, although the same heterozygous SUR1 mutation has often been found in the tested father of an affected case. The islet lesion from such cases is focal and mono- or oligoclonal based on 11p LOH (252). d. Molecules. The SUR1 gene subfamily is small and homologous to the transport ATPases. SUR1 encodes the largest subunit of the ATP sensitive potassium channel of the -cell; it is the site of sulfonylurea binding. Kir6.2 encodes the other principal channel subunit (249, 253). This inward rectifying potassium channel has a pore component (Kir6.2) and a dissimilar regulatory component (SUR1) with stoichiom-

19 Marx and Simonds Hereditary Tumors Endocrine Reviews, August 2005, 26(5): etry of four subunits of each in one pore. Cases with homozygous inactivation of SUR1 or Kir6.2 show total resistance to sulfonylureas and diazoxide, reflecting absence or disruption of the target of either drug. GK encodes glucokinase, which functions as a glucose sensor (254). GLUD1 encodes the mitochondrial glutamate dehydrogenase (GlnDH) enzyme. It catalyzes the oxidative deamination of glutamate to -keto-glutarate. A direct effect of its mutation impairs ammonium detoxification in the hepatocyte and causes systemic hyperammonemia as a unique feature with this variant of PHHI. V. Relations of Monoclonal and Polyclonal Components of Hyperfunction A. Steps in monoclonal evolution 1. Definitions and basic concepts about mutations. Genes predisposing to monoclonal or polyclonal endocrine hyperfunction can be altered on an inherited or an acquired basis. An inherited mutation is present in the zygote, and is called a germline mutation because it will be propagated after birth in all germ cells including in later generations of conceptuses. After it is transmitted from the zygote, all nongerminal cells receive it also. Whether or not an individual inherits a tumor-predisposing gene mutation, the same or other genes can be mutated at any point after birth in one or a small number of nongermline (i.e., somatic) cells. Such somatic or acquired mutation can be promoted by external factors (radiation or exposure to carcinogens) and/or inherent cellular abnormalities such as errors in DNA replication. In a hereditary syndrome involving a tumor suppressor gene, tumor expression usually follows a postnatal delay. Part of this postnatal delay represents the time necessary to acquire a somatic mutation in the previously normal copy of the same gene affected by the germline mutation, resulting in biallelic inactivation of that gene in a susceptible cell. Additional postnatal delay reflects the time, after biallelic tumor suppressor gene inactivation, to express the overgrowth of a monoclonal tumor. One reason for additional delay is that single-gene or even whole-chromosomal alterations at other loci may be necessary steps for tumor progression. A mutation that impairs the expression or activity of a tumorigenic gene product is said to cause loss of function or inactivation, (Table 4) and such a gene is classified as a tumor suppressor if it also causes loss of cell accumulation. Gain or loss of function can be shown physiologically if an in vitro or in vivo assay for the function of the encoded protein in question is available. Even if the function of the native gene product is unknown, certain mutations point to the direction of the mutation s effect; in particular, nonsense or stop codons, splice-junction mutations impairing mature transcript expression, or early truncation/frameshift mutations cause truncated protein or no protein to be expressed. Mutation that enhances or amplifies the function of a gene product are said to cause gain of function or activation, and such a mutant tumorigenic gene is classified as an oncogene if it also causes increase of cell accumulation (Table 4). If the function of the wild-type protein is unknown or if no assay is available, then gain of function might be tentatively inferred from gene amplification in tumor or from a pattern of missense mutations clustering at one or a few codons. [Note that in this context overexpression of a dominant negative mutant (see Section IV.E.1) would cause an incorrect inference.] Many missense mutations that affect only one or a few codons or small insertions that preserve the protein-reading frame may have no detrimental overall effect on protein structure. Missense mutation in disease-causing genes may inactivate or activate a protein domain and disrupt a subset of interactions without affecting other domain(s). This can even be reflected in a distinct disease phenotype associated with certain mutations; examples include differences in RET mutation associated with MEN2A vs. MEN2B (see Section IV.C.2) (Fig. 1), and phenotypic differences among some VHL mutations (255). TABLE 4. Characterization of illustrative mutations as gain of function or loss of function at level of the encoded protein Upstream interactions Gain of function (disease) Loss of function (disease) Downstream results; comments Ca 2 as agonist ligand CASR (Hypoparathyroid) CASR or CASR (FHH or NSHPT/atypical hyperparathyroid) Main regulator of the parathyroid cell. Opposite calcium effects on calcitonin from C cells. TSH as agonist ligand TSHR (Hyperthyroid) TSHR (hypothyroid) One of the main regulators of the thyroid cell. LH/HCG as agonist ligand LHR (High testosterone) LHR (deficient sex steroids) High or low sex steroids affect development and fertility. GDNF and other possible ligands at GFR- 1 4 RET (MEN2 variants) RET (Hirschsprung) Different tissues express susceptibility to gain or loss of function of RET. GPCR GNAS (McCune-Albright) PRKRA1A (Carney complex) Both mutations activate in part the same distal path including PK-A. Differences can be in Glucose-sensing or insulin-secreting Glucose-sensing GLUD1 GK (PHHI) GK Kir6.2 (PHHI) SUR1 Kir6.2 (PHHI) GK or GK Kir6.2 (diabetes mellitus) tissue targets, effector, or cell compartments. Appropriate direction of mutation in any of four genes yields similar phenotype of PHHI. Central protein in control of insulin release (in response to blood glucose). Ultimate phenotype depends equally on direction of the mutation effect (gain or loss in activity of the mutant protein) and on the context (promoting or inhibiting secretion) in which the encoded protein functions. HCG, Human chorionic gonadotropin; GDNF, glial-derived neurotrophic factor; GFR, GDNF family receptor;, gain of function;, loss of function (heterozygous);, loss of function (homozygous).

20 634 Endocrine Reviews, August 2005, 26(5): Marx and Simonds Hereditary Tumors Mutations that enable the mutant protein to bind and sequester the normal copy of the protein or other protein partners in a nonproductive fashion and thereby impair the function of the wild-type allele are termed dominant-negative or dominant-interfering. In such cases the single mutant allele imparts a loss-of-function phenotype in the cell. For example, the dominant-negative effect of certain p53 missense mutations in sporadic and Li-Fraumeni-associated tumors has also been demonstrated in transgenic mice (256). The p53 tumor suppressor normally functions as a tetramer, and its missense mutants likely impair the function of wildtype p53 protein via oligomerization together with normal copies. In principle, the frequent nonsense mutations that encode truncated versions of a gene product might create dominantnegative proteins containing intact N-terminal domain(s). More often, however, nonsense mutants are not expressed and cause disease by complete loss of encoded protein. Truncations often severely impair the folding and stability of the encoded protein, and such mutants are rapidly degraded. Furthermore, eukaryotic cells possess machinery for RNA surveillance to identify some nonsense mutation-containing transcripts and target such mrna for degradation (257). 2. Knudson s two-hit mechanism. A model for tumor development that eventually accounted for the interaction of many inherited and somatic gene mutation events was proposed by Alfred Knudson from his epidemiological analysis of retinoblastoma (258). Retinoblastoma is much more common in sporadic than familial settings; in particular, familial cases had special differences including much earlier age of onset and more frequent bilaterality. The two-hit hypothesis of neoplasia suggests that two events or hits conferred a selective growth advantage to one affected cell as a prerequisite for monoclonal progression. It is now well established that in many hereditary tumor cases the first event is a mutation in the germline DNA and therefore present in all the cells of the affected offspring. The early age of onset of familial tumor cases and the tendency for multifocal disease are each explained by the greater likelihood of a second somatic event ( second hit ) arriving in a field where all cells already have the first hit. Although the two-hit hypothesis does not require that both hits affect the same gene, both copies of the same gene are virtually always inactivated in tumors involving tumor suppressor genes. The second hit, inactivating the wild-type allele in tumor suppressor gene-associated tumors, is most often a rearrangement or large deletion, typically as large as an entire chromosome copy. Point mutation or epigenetic changes at the same locus may rarely be the second hit and then not associated with subchromosomal LOH. If a sporadic tumor arises entirely from events at other genes, any comparison of expressions to the two-hit tumor would be unpredictable (55). 3. Two hits at an oncogene. The two-hit model of tumorigenesis is relevant not only for tumor suppressor genes such as RB1 or MEN1, but also for several oncogenes at which the two hits seem to function similarly to enhance stepwise gain of function of the gene product. In a study of tumors from patients with hereditary papillary renal carcinoma, involving germline missense mutation of the MET oncogene, a second hit was identified as nonrandom duplication of the chromosome 7 allele containing the mutant MET allele (259). Examples of two activating hits are also seen in several MEN2-associated tumors involving the MET-related RET oncogene as follows: Pheochromocytomas from patients with MEN2 have a first hit identified as the germline gain-of-function RET missense mutation; a second hit is most frequently duplication of the mutant RET allele resulting from trisomy of chromosome 10. A distinct second-hit mechanism was identified in TT cells derived from MEN2-associated MTC (260). These cells were found to have tandem duplication of the mutant RET allele on one chromosome 10. Somatic point mutation of the wild-type RET allele has also been found as the second hit in the setting of MEN2A and FMTC. In particular, MTC tumor from a patient with early-onset disease in the setting of FMTC and germline Val804Leu RET mutation harbored a somatic activating Met918Thr mutation involving the wild-type allele (54). MTC in two families with a germline Val804Met or Ala883Thr RET mutation was associated with the homozygous but not with the heterozygous RET germline mutation (95, 261). Less commonly, there is loss of the wild-type RET allele (262). Also, loss of the wild-type RET allele due to a large intragenic deletion documented by LOH analysis was seen in a MTC tumor metastasis in a patient with germline RET mutation (263). It is likely that each of these seemingly different second hits at an oncogene further increases the overall expression of this oncogene. Note, however, that two hits at an oncogene cause quantitative changes, whereas two hits at a tumor suppressor gene cause an all-or-none change. And the frequency of a second hit at an oncogene locus is not known. 4. More than two hits. The evolution of endocrine tumors often requires more than two hits. In fact there are no tumors where two hits have been proven sufficient to cause monoclonal overgrowth (264). The stepwise accumulation of more than two hits has been best characterized in the evolution of colon cancer, which requires the biallelic loss of function of several tumor suppressors and gain of function of one or more oncogenes (265). The similar role of multiple genes in the monoclonal progression of certain endocrine neoplasias has been suggested by tumor analysis for LOH, comparative genomic hybridization, or gene mutation. There is evidence, for example, of at least three defined hits in a subset of MEN2A-related pheochromocytomas (170). In addition to the germline RET mutation in these tumors, biallelic inactivation of the VHL gene at 3p25 was documented by identification of VHL point mutation in the retained allele in three out of four tumors studied. 5. Epigenetic silencing, including loss of imprinting. Epigenetic events are not hereditary but can be established at one allele early in development and can have lasting effects on the structure of chromatin or the pattern of DNA methylation.

21 Marx and Simonds Hereditary Tumors Endocrine Reviews, August 2005, 26(5): The outcome can be gain or loss of gene function. Such an epigenetic change can provide a necessary hit resulting in tumor initiation and/or progression, particularly when it occurs in combination with modification at the other allele (266). Epigenetic changes can contribute to tumorigenesis such as with the inappropriate and pathological hypermethylation of cytosine residues in CpG islands in 5 promoter regions of tumor suppressor genes leading to transcriptional silencing (267). Epigenetic silencing is frequent and particularly well documented with VHL gene-associated tumors both in the hereditary and sporadic setting (266). Hereditary paraganglioma types PGL1 and PGL2 (associated with SDHD and SDHC mutation respectively) are expressed only from paternal inheritance because either gene on chromosome 11 is imprinted and not expressed from the maternal allele ( ). Interestingly, some of these paragangliomas show LOH at the SDHD locus at 11q23, with the consistent finding that loss is restricted to the maternal (wildtype) allele (268, 271, 272). Loss of gene imprinting may also contribute to tumorigenesis as is seen in WT2-associated Wilms tumor. Whereas WT1 at locus 11p13 is a tumor suppressor gene, the putative tumor suppressor gene at the WT2 locus on 11p15 has not been identified. Instead, the WT2 locus holds a cluster of several important imprinted genes including IGF-II, H19, and P57/KIP2. IGF-II encodes an embryonal growth factor that is normally expressed only from the paternal allele. Loss of imprinting (LOI) of IGF-II has been found in some Wilms tumors; this results in abnormal expression from the maternal allele (due to duplication on the maternal allele of the paternal pattern of epigenetic modifications) and overexpression of the growth factor due to its abnormal biallelic expression (273). Two other genes located at 11p15 may also contribute to tumor formation in Wilms tumor due to LOI. H19 and P57/KIP2 are normally expressed solely from the maternal allele and have growth suppressive properties (regarding p57/kip2, see Section V.E.1.b and Fig. 2). The LOI in Wilms tumor represents an altered pattern of DNA methylation that results in duplication of the paternal pattern of epigenetic modifications on the maternal allele. The resulting abnormal biallelic methylation of the H19 promoter causes a pronounced decrease in H19 (274) and P57/KIP2 (275, 276). Another possible example of LOI promoting tumor development is seen in the pathogenesis of acromegaly-associated pituitary somatotroph tumors. Some 40% of such tumors harbor somatic activating mutation in GNAS encoding the gsp oncoprotein. GNAS is biallelically expressed in most tissues but in the normal pituitary GNAS is expressed solely from the maternal allele (277). Although the vast majority of gsp-positive somatotroph tumors demonstrate mutation of the maternal allele, relaxation of imprinting with biallelic expression of GNAS is often seen, suggesting such LOI may also contribute to tumorigenesis (277). 6. Caretakers and gatekeepers. Most tumor suppressor genes have been classified in one of two broad categories of function, namely caretakers and gatekeepers (278). Certain tumor suppressor genes play a general role in maintaining chromosomal stability, and when disrupted they allow a variety of secondary mutations in other genes through a process of generalized genomic destabilization. Such genes have been called caretakers of the genome because their normal role is to maintain the integrity of all chromosomes (279). Biallelic inactivation of a caretaker tumor suppressor gene is, in and of itself, insufficient for tumorigenesis, and subsequent steps such as somatic mutation of additional tumor suppressors in susceptible tissues are usually required for tumor initiation and/or progression (279, 280). Mutation in caretaker tumor suppressors can transmit a dominant susceptibility to a familial cancer syndrome, such as familial breast and ovarian cancer from BRCA1 or BRCA2 mutation. In these cases, the second hit results from a somatic mutation of the same gene. A recessive inheritance pattern is less common among familial tumor syndromes and involves tumor suppressor genes such as in Bloom syndrome from BLM mutation and in Werner syndrome from WRN mutation. The second category of tumor suppressors is genes that directly control growth or death in selected tissues and are thus termed gatekeepers. Inheritance of germline inactivat- FIG. 2. Distribution of germline mutated genes or their encoded proteins among signaling pathways and their associations with hormone excess syndromes.

22 636 Endocrine Reviews, August 2005, 26(5): Marx and Simonds Hereditary Tumors ing mutation in a gatekeeper tumor suppressor gene is associated with relatively tissue-specific family cancer syndromes with prominent development of tumors in one or a few tissues. Somatic mutation in a gatekeeper gene such as MEN1 is also common in sporadic tumors of the same tissues as those involved in the familial syndromes. Mutations of caretaker tumor suppressor genes, in contrast, are uncommon in sporadic tumors, probably because biallelic inactivation of the caretaker gene and the subsequent mutation of rate-limiting gatekeeper genes would be required in the same cell for monoclonal expansion (259, 262). Another property, shared between oncogenes and the gatekeeper class of tumor suppressors, is that sporadic tumors of tissues typically affected in certain hereditary syndromes of cancer susceptibility frequently contain mutations of the same genes as in familial tumor. For example, sporadic MTC has somatic RET mutation in approximately 25% of tumors. There are exceptions to the generalization that only gatekeeper-type tumor suppressors contribute substantially to the etiology of sporadic tumors of the same type as seen in the associated familial syndrome. For example, caretakers of the genome include the DNA mismatch repair genes hmsh2 or hmlh1, implicated in a tissue-selective way in most cases of hereditary nonpolyposis colorectal cancer. All or most oncogenes can be classified as gatekeepers because, like many tumor suppressors, they directly control the rate of cell birth or the rate of cell death (281, 282). There are too few such examples of familial cancer syndromes due to germline mutation of protooncogenes to allow broad generalizations. Oncogenes were first identified as dominantly acting transforming genes, and together with their mutation only a small number of additional hits seem to be required TABLE 5. Monoclonal and polyclonal states with hereditary hypersecretion to initiate tumor formation, such as in the examples of mutant RET or MET cited above (259, 262). B. Polyclonal features within syndromes of hormonal excess 1. Polyclonal hormonal excess as endpoint in a major category. Several hereditary disorders of hormone excess result from diffuse hyperfunction of the affected tissue without monoclonal tumor formation. Such syndromes typically affect a single endocrine tissue (Table 5). Diseases such as FHH due to heterozygous inactivation of the CASR and nonimmune hyperthyroidism (hereditary toxic thyroid hyperplasia) due to gain-of-function mutation of the TSHR gene were discussed above. In contrast to sporadic and hereditary monoclonal neoplasms that require two or more hits over one or several decades to develop, polyclonal syndromes of hormonal hyperfunction are essentially one-hit processes, expressed in an all-or-none fashion, with hormonal hyperfunction present in all secretory cells of affected tissues and often evident from birth (Table 5B) (167). This polyclonal paradigm applies also to some syndromes involving homozygous mutant genes such as CASR in NSHPT, and SUR1 or Kir6.2 in PHHI. The regulatory or set-point disturbance in syndromes of polyclonal hormonal excess can be the principal cellular expression and can sometimes be associated with little or no hyperplasia by histology. This is true for example in FHH in which parathyroid gland size and cellularity can overlap substantially with normal (218, 219). Usually the systemic hormone regulatory defect in syndromes of polyclonal hormonal excess can even be expressed through very few affected cells; thus, partial removal of the affected glandular tissue frequently results in persistent disease. Total ablation of the hyperfunctioning gland or glands A. Classification of some states (mostly with known mutant gene) based on whether their hormone overproducing glands are clonal or polyclonal Principal overactive tissue Syndrome with monoclonal hypersecretion Syndrome with polyclonal hypersecretion Parathyroid gland MEN1, HPT-JT FHH, NSHPT Gonadal androgenic cells McCune-Albright, Li-Fraumeni, Beckwith-Wiedemann, Testotoxicosis Carney complex Non C cell thyrocyte Cowden, APC, Werner, Carney complex Nonimmune hyperthyroidism Islet insulin or somatostatin cells TSC, NF1, MEN1 PHHI (4 genes) Anterior pituitary Familial isolated pituitary, MEN1, Carney complex, None McCune-Albright Chromaffin tissue MEN2, PGL1 3, etc., VHL, Carney triad, MEN1, NF1 None Adrenal cortex Carney complex, MEN1 None B. Hereditary hormone excess: clinical properties according to gland classification as monoclonal or polyclonal (see Table 5A) Parameter for comparison Monoclonal hormonal excess Polyclonal hormonal excess Age of onset for hormone excess Delay after birth At birth or in utero Hyperfunction in multiple tissues Usual Rare a including nonendocrine Penetrance in affected tissue Variable Similar and 100% Endocrine gland macropathology Asymmetric enlargement Symmetric enlargement; or normal size Endocrine gland micropathology Focal hypercellular; maybe nodules Diffuse hypercellular; no nodules Endocrine tissue malignancy Inherent Rare Response to subtotal ablation Remit; may recur Persist Pathophysiology Somatic steps at second allele and beyond Germline mutation causes the full phenotype in syndromal tissue Modified from Marx, 1999 (167). a Rare, Not an intrinsic feature of the syndrome but may rarely occur as an unrelated or random feature.

23 Marx and Simonds Hereditary Tumors Endocrine Reviews, August 2005, 26(5): by surgical or medical means, with appropriate replacement therapy, is often the only successful treatment (217, 283). 2. Hyperplasia as a contributor to polyclonal or monoclonal hormone excess a. Polyclonal. Hyperplasia is an increase in cell numbers, and it is often identified through histology. Because the glandular hyperplasia sometimes seen in certain syndromes of hormonal excess is diffuse and without nodule formation, it is presumed that all of the expressing cells exhibit the phenotype and that the associated hyperplasia represents a polyclonal process. This presumed polyclonality of hyperfunctioning hormonal cells has not been rigorously addressed in any of these syndromes, mainly because this distinction is beyond current methods (167). There is evidence in several endocrine tissues that genes associated primarily with polyclonal hyperfunction may also contribute to monoclonal neoplasia in a nonhereditary setting. Patients with autosomal dominant nonimmune hyperthyroidism due to heterozygous gain-of-function mutations in the TSHR gene have diffuse goiters in a presumed polyclonal process that typically does not generate thyroid nodules or cancer (233). Even so, similar activating mutations in TSHR are found in sporadic toxic thyroid adenomas and, rarely, in differentiated thyroid cancer (284, 285); both are monoclonal. Although Leydig cell adenoma or testicular cancer has not been reported with familial male-limited precocious puberty (235), some sporadic Leydig cell tumors demonstrate similar activating LHR mutation, suggesting that this DNA change can be permissive if not sufficient for monoclonal transformation (237). b. Precursor to monoclonal overgrowth. Hyperplasia may also precede monoclonal progression associated with genes predisposing to hereditary monoclonal hormonal excess. Histological hyperplasia may coexist with monoclonal or polyclonal accumulation of cells. In MEN syndromes, for example, hyperplasia may precede tumor development in a subset if not all of affected tissues. By histological criteria, multifocal C cell hyperplasia is a precursor to the development of MTC in MEN2 (287, 288). Surprisingly, analysis of X-chromosome methylation patterns in microdissected foci of C cell hyperplasia suggested that monoclonality had been established even before this early stage in C cells within the MEN2 thyroid (169). A transgenic mouse model of MEN2A also demonstrates early, bilateral C cell hyperplasia that precedes MTC development (170). Diffuse and nodular adrenal medullary hyperplasia is also a frequent precursor of pheochromocytoma in MEN2 in humans (289) and in a transgenic mouse model (290). Interestingly, such chromaffin cell hyperplasia is not a histological feature of VHL-associated pheochromocytoma (177). A mouse model of MEN1 allowed prospective histological analysis of target endocrine tissues in Men1 / mice (153). The Men1 / mice sequentially developed medium-sized hyperplastic pancreatic islets, larger focally dysplastic islets, and then small islet cell tumors that preceded the development of larger, numerous islet tumors. Dysplastic pancreatic islets and islet cell tumors developed at an even younger age in a mouse insulinoma model in which homozygous loss of Men1 was forced in pancreatic -cells (291). The hyperplastic lesions in unconditional Men1 knockout model showed two criteria that Men1-associated islet -cell hyperplasia was polyclonal; these were retained heterozygosity at the Men1 locus and retained expression of menin protein (see Section V.E.2). 3. Other haploinsufficiency processes. Inheritance of a single mutant allele of a tumor suppressor may in itself produce a phenotype in certain tissues, representing a mechanism different from the tumor initiation that would normally require somatic inactivation of the normal allele in a susceptible cell. Gene dosage, and particularly haploinsufficiency, phenotypes are by definition one-hit processes, and examples can be found in a variety of inherited neoplasia syndromes. Such terminology will accommodate both a one-hit loss of function (haploinsufficiency) and a one-hit gain of function, as with TSHR, LHR, GK, and GLUD1. Haploinsufficiency of tumor suppressor genes in some tissues may provide a broad substrate for subsequent hits at other loci leading to monoclonal transformation. In the setting of an inherited neoplasia syndrome, haploinsufficiency of certain tumor suppressors appears sufficient for tumor initiation and/or progression, because some such tumors don t demonstrate LOH. Whether tumorigenesis in tissues that don t demonstrate LOH at a tumor suppressor locus might involve inactivation of the normal allele by another mechanism, and thus reflect an occult monoclonal process, has not been adequately excluded in most of the studies below. For example, determination of tumor suppressor protein expression in such tumors, which might resolve this question, is largely lacking. Although the principal tumor types in MEN1 are usually associated with somatic inactivation of the normal allele and demonstrate LOH at the MEN1 locus at 11q13, haploinsufficiency may contribute to certain MEN1-related neoplasms. Thymic carcinoids in MEN1 are frequently malignant and do not demonstrate LOH at the MEN1 locus (292, 293). Adrenocortical enlargement in MEN1 patients is also not usually associated with LOH at the MEN1 locus (294, 295). Haploinsufficiency of P53 in the Li-Fraumeni syndrome may support monoclonal transformation of many affected tissues. Approximately half of the tumors studied from patients with germline P53 mutation and the Li-Fraumeni syndrome retain the wild-type allele (296, 297). Tumor development in a P53 / mouse model of Li-Fraumeni syndrome also occurs frequently with retention of the wild-type allele (298). Haploinsufficiency of NF1 seems to play a key role in the pathogenesis of several mainly monoclonal expressions of NF1 (299). Melanocytes isolated from café-au-lait macules from NF1 patients retained heterozygosity at the NF1 locus, suggesting haploinsufficiency (300). The neurofibromas in NF1 are admixtures of several cell types, including Schwann cells, fibroblasts, and mast cells. In mouse models, Nf1 nullizygous Schwann cells can form tumors, but full expression of the complex neurofibromata characteristic of NF1 also requires the participation of Nf1-haploinsufficient mast cells that interact with the Schwann cells, presumably through the elaboration of cytokines (198). Some other polyclonal macroscopic expressions of hered-

24 638 Endocrine Reviews, August 2005, 26(5): Marx and Simonds Hereditary Tumors itary tumor syndromes may also result from haploinsufficiency of the relevant gene. The macrocephaly associated with germline PTEN mutation in Cowden syndrome and the related Bannayan-Riley-Ruvalcaba syndrome can be seen at birth without evident monoclonal proliferation and is therefore likely a consequence of PTEN haploinsufficiency (301). The macrocephaly in human syndromes associated with PTEN mutation may reflect abnormalities in neural differentiation such as those identified in a mouse model where PTEN haploinsufficient neural progenitor cells exhibited increased migration and proliferation and were more resistant to apoptosis (302). Congenital tibial dysplasia and other skeletal manifestations of NF1 may be expressed in early childhood and are a likely result of NF1 haploinsufficiency (303). C. Steps in histopathology different from or beyond hyperplasia 1. Cystic features. The abnormal cystic histology in various tissues from patients with selected hereditary neoplasia syndromes may provide a clue to disease pathophysiology. Monoclonal renal and hepatic cysts were first shown to develop by a two-hit mechanism with LOH at the PKD1 locus but without parallel tumor in hereditary polycystic kidney disease ( ). Monoclonal renal cysts also are likely to represent a precursor or parallel process in the development of renal cell carcinoma (RCC) in some familial cancer syndromes including tuberous sclerosis and VHL. RCC is sometimes a manifestation of tuberous sclerosis associated with either TSC1 and TSC2-linked disease (307, 308). The Eker rat model of tuberous sclerosis with TSC2 mutation exhibits both polycystic kidney disease (309) and RCC; the latter also express abnormal renal tubules that exhibit LOH at the TSC2 locus (310). Most cases of tuberous sclerosis with severe polycystic kidney disease also have large deletions involving TSC2 and the adjacent PKD1 (311). Nevertheless, renal cysts may be an intrinsic feature of pure tuberous sclerosis because some patients with intragenic deletions confined to TSC2 also have significant renal cystic disease (311), and patients from families with linkage to TSC1 on chromosome 9 also manifest renal cysts (307). Multiple renal cysts and RCC are also common in VHL (312). Benign and atypical renal cysts in VHL involve clear cells and exhibit loss of the wild-type VHL allele in the vast majority of microdissected lesions and thus may represent a monoclonal precursor to RCC (313). VHL patients also develop multiple pancreatic cysts that are likely monoclonal precursors to the development of microcystic serous adenomas. The microdissected cysts, like the microcystic serous adenomas, uniformly demonstrate LOH at VHL, involving the wild-type allele (178). Although an association with RCC has not been established, HPT-JT expresses bilateral renal cysts as well as renal hamartomas, the latter of which show LOH at the HRPT2 locus (105); cystic parathyroid tumors are also associated with HPT-JT (315). Cystic tumor of the endocrine pancreas is uncommon, and usually implies MEN1 (316). The pancreas of MEN1 cases also contains small collections of pluripotent monoclonal cells that could represent islet tumor precursors or an alternate expression from monoclonal cells (317). 2. Benign vs. malignant monoclonal neoplasm. An interesting observation in patients with germline mutation in a tumor suppressor gene is the reproducible and tissue-specific variation in the malignant potential of syndromic tumors. In MEN1, for example, certain characteristic tumors, like facial angiofibromas and monoclonal parathyroid tumors, are highly penetrant yet almost universally benign, although MEN1-associated enteropancreatic and foregut carcinoid tumors are often malignant (318). In VHL, one of the most penetrant tumors, retinal angioma, is benign although the highly morbid renal tumors are almost always clear cell cancers (312). The fibro-osseous jaw tumors of HPT-JT are characteristically benign despite the high frequency in this syndrome of parathyroid cancer (see Section IV.B.1). Current models do not adequately explain these observations, although presumably tissue-specific variation in the expression of other factors with overlapping function could protect certain tissues from malignant transformation despite biallelic loss of a given tumor suppressor. A corollary finding is that despite the overlapping involvement of a particular endocrine tissue in different syndromes, mutations in certain genes, but not others, have strong association with malignant transformation. The uniquely high incidence of parathyroid cancer in HPT-JT (15%) relative to other forms of familial and sporadic primary HPT ( 1%) indicates that HRPT2 encoding parafibromin (76) plays a key role in malignant transformation of parathyroid tumors. This association is supported by analyses of apparently sporadic parathyroid cancers that usually show small intragenic mutation in both alleles of HRPT2 (107, 319). For example, contrast the neoplastic expression of HRPT2 mutation in parathyroid with that of MEN1 mutation. Despite the early onset, involvement of multiple glands, and frequent recurrence, parathyroid tumor associated with germline MEN1 mutation is almost always benign (318). Malignant transformation of other endocrine tissues may also be strongly associated with mutation in other specific genes. The strong association between RET gain-of-function mutation and familial and sporadic forms of MTC was discussed above, as was the association of RET/PTC and papillary thyroid carcinomas (320). Mutation in P53 is found in 70% of sporadic adrenocortical cancers but not in benign adrenal tumors (134). The association between P53 and adrenal cancer is further supported by the observation that some patients with Li-Fraumeni syndrome and germline mutation of P53 show early expression of this rare endocrine malignancy (130). A related finding is that certain mutations in a tumor susceptibility gene may predispose to a more aggressive neoplastic phenotype. As previously noted, the germline methionine-918 mutations in RET are associated with highly aggressive and early-onset MTC in the MEN2B syndrome. Among sporadic MTC, the identical mutation in somatic DNA is also associated with more aggressive tumor (53, 54). Mutation at LHR codon 578 is also associated with a more severe clinical phenotype, which may include frank Leydig cell adenoma instead of polyclonal testicular involvement

25 Marx and Simonds Hereditary Tumors Endocrine Reviews, August 2005, 26(5): associated with the mutation of other LHR codons in the germline (237). 3. Differentiated functions in monoclonal tissue. Endocrine neoplasms in the setting of a hereditary syndrome of hormonal excess show abundant evidence of conserved differentiated function. Many such tumors clearly retain the ability to synthesize, store, and release hormone, and also frequently maintain some aspects of correct responsiveness to normal regulatory stimuli. Indeed these properties are central to their classification as hormonal; through other molecules in differentiation, they may be responsive to regulators and to related tumor treatment. Prolactinomas in the setting of MEN1, like sporadic prolactinomas, are generally sensitive to D2 dopamine receptor agonists providing the basis for the main medical therapy (321). Although parathyroid tumors have decreased expression of the cas-r (322), benign and malignant parathyroid tumors can respond to calcium and also to calcimimetic drugs, with either acting through the cas-r (323, 324). Sporadic papillary thyroid cancer retains the ability to take up and retain iodine, providing the basis for radioiodine therapy. The ability to take up catecholamine precursors and concentrate them in synaptic storage vesicles is retained in VHL- and MEN2A-associated pheochromocytomas; this provides the basis for tumor localization by [ 131 I]- metaiodobenzylguanidine scintigraphy and positron emission tomography with 6-[ 18 F]fluorodopamine or 5- hydroxytryptophan (325, 326). D. Relations of tumorigenic germline mutation to sporadic tumor 1. Contributions to sporadic monoclonal neoplasia. As emphasized above, many of the genes first identified for their etiological role in a monoclonal or polyclonal syndrome of hereditary hormonal excess were later recognized to be important in a variable fraction of sporadic tumors in the same affected tissues (Table 1). This is particularly true for the gatekeeper category of genes implicated in hereditary monoclonal tumors. Genes implicated by mutation and/or epigenetic change also in sporadic neoplasms include the following: MEN1, HRPT2, RET, VHL, PTEN, P53, and GNAS (see Sections IV.A IV.C). Less frequently, genes implicated in polyclonal syndromes of hereditary hormonal excess play a role in sporadic tumorigenesis. This applies to TSHR and LHR but so far not to CASR or any of the four genes causing PHHI (see Section IV.E). 2. Genes mutated in sporadic but not hereditary tumor. Several well-studied oncogenes and tumor suppressors implicated in sporadic benign or malignant tumors have not been associated with a hereditary syndrome; this applies to oncogenes such as mutationally activated GTPases (GNAS, H- RAS, N-RAS, and K-RAS) implicated with variable frequency in many sporadic tumors. One explanation for this has been that constitutive G protein signaling in most tissues by such a GTPase with many important downstream regulatory targets would probably disrupt embryogenesis (327). Constitutive expression of many oncogenes representing mutated cell surface growth factor receptors and nonreceptor tyrosine kinases would also be likely to severely impair embryogenesis for similar reasons. For example no hereditary syndrome involving EGFR or SRC has been identified, despite their frequent involvement in sporadic squamous cell and colon cancer, respectively, and mutations of several tyrosine kinase genes have been implicated only in sporadic tumors (328, 329). In this light it is perhaps surprising that the MET and RET oncogenes, which each encode a mutant tyrosine kinase receptor, can transmit familial cancer syndromes (hereditary papillary renal carcinoma and MEN2A/MEN2B/FMTC, respectively; see Section IV.C.2) with a normal conceptus. The compatibility of germline mutations of these oncogenes with seemingly normal embryogenesis may reflect in part their restricted tissue expression patterns. During development, for example, RET expression is confined largely to the nervous and urogenital systems (330). The compatibility of RET germline oncogenic mutation with embryogenesis may also reflect the complexity of the multimeric cell surface complexes in which it functions. This is because restricted tissue expression of one or more among a family of homologous glial-cell-derived neurotrophic factor ligands and/or glycosyl phosphatidylinositol-linked coreceptors with which ret normally complexes might limit productive signaling via ret and blunt the impact of an activating mutation (331). Besides these oncogenes, there are examples of tumor suppressors important for development of sporadic tumors that have not been implicated in a hereditary syndrome (Fig. 2). Biallelic loss of P15 (INK4B), encoding an important cell cycle checkpoint regulatory protein (see Section V.E.1 and Fig. 2), is seen in sporadic tumors such as anaplastic meningiomas (332), gliomas (333), and hematological malignancies (334), although no hereditary syndrome of tumor predisposition involving this gene has been reported. Speculations similar to those for oncogenes would allow that haploinsufficiency of such a gene or an embryonic second hit could be lethal to embryogenesis. 3. Large chromosomal or subchromosomal deletion is common with some somatic mutations but rare in germline mutations. The LOH of tumor DNA at a tumor susceptibility locus usually reflects a large somatic deletion in one copy of that chromosome, which can reflect loss of a gene (if the LOH is only with intragenic markers), loss of a gene with adjacent genes, or even loss of an entire chromosomal copy. This event frequently constitutes the second tumorigenic hit at the locus and usually involves loss of the entire wild-type copy of the gene. In contrast, germline mutations in inherited cancer syndromes are most often small insertions, deletions, or point mutations. The rarity of large germline deletions is most likely because of reduced germinal cell and embryonic viability associated with large subchromosomal or entire chromosomal deletions involving many adjacent genes. During gametogenesis, meiotic checkpoints select against passage of large deletions and rearrangements through the germline. Early in meiosis, pairing of maternal and paternal chromosomes in germ cells is followed by synapsis and crossing over of chromosome segments in the process of homologous recombination. Large chromosomal deletions impair this carefully regulated process and can lead to germ cell apoptosis through the action of the meiotic recombina-

26 640 Endocrine Reviews, August 2005, 26(5): Marx and Simonds Hereditary Tumors tion, or pachytene, checkpoint (335). The pachytene checkpoint prevents meiotic nuclear division in cells that fail to complete chromosomal synapsis, as would occur when large segments of a chromosome are unpaired (336), and prevents the production of aneuploid gametes. Apart from checkpoint activation, large chromosomal deletions would be expected to cause haploinsufficiency of hundreds of genes. Such largescale haploinsufficiency may in itself be detrimental to critical meiotic processes or other processes for cell survival, although specific studies are lacking. In contrast to the deleterious effects of large chromosomal deletions, point mutations, small deletions, or insertions in germ cell DNA have no adverse impact on synapsis, meiosis, or subsequent events. How can the process of mitosis sometimes tolerate or even be promoted by large somatic subchromosomal deletions or even loss of an entire chromosome as occasionally seen in tumor cells? There is no crossing over in mitosis and therefore no equivalent of the pachytene checkpoint in somatic tissues. The spindle checkpoint in mitosis is insensitive to large subchromosomal deletions that don t involve the centromere, because its main function is to ensure attachment of every centromere to the spindle apparatus (337, 338). Somatic loss of an entire chromosomal copy usually results from nondisjunction defects during chromosomal segregation and may be propagated to daughter cells during monoclonal expansion of a tumor. DNA damage activates mitotic checkpoint kinases, such as ATM and ATR (ATM- and Rad3- related kinase), that primarily respond to specific types of DNA damage, such as the presence of single-stranded DNA or double-stranded DNA breaks, or to altered chromatin structure (339). It may be that loss of an entire chromosome fails to activate such mitotic checkpoint kinases. Loss of the entire chromosome can also be masked by duplication of the retained copy of the chromosome, allowing bypass of both DNA damage and spindle checkpoints. Such gene conversion, i.e., somatic recombination resulting in duplication of the missing portion of the chromosome, may result in whole chromosome homozygosity or at least a region of isodisomy spanning the affected area (340). The frequency of this compensation for chromosomal loss is not certain (341). Haploid gametes have no such compensatory option. Interestingly, a nonquantitative analysis of polymorphic microsatellite markers in the region of gene conversion would show LOH just as if the deletion were uncompensated by somatic recombination. In contrast, the comparative genomic hybridization method is sensitive to DNA ploidy across a large zone (50 kb). Not only are many large deletions tolerated during mitosis in somatic tissues, but some deletions resulting in biallelic inactivation of a tumor suppressor gene clearly confer a growth advantage to cells. Furthermore, the sometimes deleterious effect of contiguous gene loss in such large deletions may sometimes be compensated by gene conversion. HRPT2 is remarkable in this regard. The loss-of-function event in the second or remaining wild-type allele is rarely a large DNA loss but most commonly a missense mutation (107). This may be the unusual example of a tumor suppressor locus at which a large second hit is generally lethal to the cell. E. Animal models genes acting alone or in cooperation for tumorigenesis The first oncogene implicated in the pathogenesis of parathyroid tumors was PRAD1 (later termed CYCD1) encoding cyclin D1, a key regulatory protein in control of the cell cycle. CYCD1 mutation by rearrangement has been found in 4 5% of parathyroid adenomas, and CYCD1 is overexpressed in 20 40% of sporadic parathyroid adenomas (reviewed in Ref. 342) (Fig. 2) and in other tumors, including breast cancer (343) and centrocytic B cell lymphoma (344). Defective expression of one or more cell cycle checkpoint proteins is now recognized in the action of many tumor suppressors and oncogenes important in endocrine monoclonal neoplasia (345). Targeted mutation of tumor suppressor genes and introduction of oncogenes as transgenes in rodent models has enabled the recognition of genes that do and do not interact with cell cycle regulatory proteins to promote tumorigenesis in specific tissues (Fig. 2). Mouse strains with single gene defects can be mated with strains expressing other tumorigenic defects, and the defects can be targeted to only selected tissues. Gene interactions in tumor formation can be monitored through the resulting offspring. Much of this work has been done with nonendocrine tumors. 1. Interaction with cyclin-dependent kinase (CDK) inhibitors in mouse models a. INK4 family. Mouse models demonstrate that many of the genes involved in the prb/e2f pathway that regulate progression through the G 1 phase of the cell cycle can cooperate with other genes to promote tumorigenesis (Fig. 2). The activity of the CDK4/6-cyclin D complexes during G 1 is negatively regulated by members of the INK4 class of CDK inhibitors, including p16 (INK4A), p15 (INK4B), and p18 (INK4C). The major substrate for phosphorylation by CDK4/ 6-cyclin D is prb. Because phosphorylated prb loses its ability to inhibit DNA synthesis, the normal activity of the INK4 class of CDK inhibitors is to slow progression through G 1. Although the Rb1 null mutation is embryonic lethal, mice heterozygous for Rb1 mutation develop benign tumors of the pituitary intermediate lobe with high penetrance (346). This phenotype can be enhanced by coexistent P27 mutation (see Section V.E.1.b). Inactivation of P16 (INK4A) is associated with familial melanoma (347) and familial pancreatic cancer (348); there is no known familial neoplasia syndrome involving loss of function of P15 (INK4B) or P18 (INK4C) (349). Germline missense mutation affecting the p16 (INK4A) binding domain of CDK4 has also been implicated in familial melanoma (350). Knock-in mice expressing this mutant CDK4 allele when treated with topical carcinogens demonstrate enhanced sensitivity to the development of invasive melanoma (351). Mice nullizygous for P18 (INK4C) but not P15 (INK4B) were also sensitized to development of early stage melanomas in response to carcinogen treatment (351). Haploinsufficiency of P18 (INK4C) sensitized mice treated with nitrosamines to the formation of intermediate lobe pituitary tumors and other neoplasms (352). b. Cip/Kip family. Members of Cip/Kip, a second family of CDK inhibitors (p21/waf1, p27/kip1, and p57/kip2) also interact with other cell cycle regulators and other genes to

27 Marx and Simonds Hereditary Tumors Endocrine Reviews, August 2005, 26(5): regulate tumorigenesis in mouse models (Fig. 2). Members of the Cip/Kip family of CDK inhibitors inhibit the function of both G 1 -phase cyclin D-CDK4/6 and cyclin E-CDK2 complexes as well as inhibit the G 1 /S phase cyclin A-CDK2 complexes. Earlier and more aggressive pituitary tumor formation was seen in P27 /, RB / crossbred mice than in either P27 / or RB / parental strain, indicating an interaction between these genes (353). Another positive interaction of p27/kip1, with the Apc tumor suppressor, was seen in the earlier and more aggressive gastrointestinal tumors seen in APC /, P27 / mice than in APC heterozygotes alone (354). PTEN activity induces the expression of p27/ Kip1, and highly penetrant prostate cancer with early onset resulted from cooperative interaction between the PTEN and P27/KIP1 tumor suppressor genes in crossbred mice (355). Intriguing interactions of either p27/kip1 or p21/waf1 with p18 (INK4c) were demonstrated in strains of doubly mutant mice with phenotypes suggesting overlaps of pathways in MEN1 and MEN2 syndromes (356). Mice homozygous for both P18 (INK4C) and P27/KIP1 deletion, as well as mice nullizygous for only three of the four P18 (INK4C) and P27/KIP1 alleles, developed tumors and/or hyperplasia of multiple tissues including pituitary, thyroid C cells, adrenals, and parathyroid. The doubly nullizygous P18 (INK4C) and P21/WAF1 mice developed pituitary tumors and gastric neuroendocrine hyperplasia (356). This supported the possibility that MEN1 and MEN2 could include some identical CDKIrelated downstream expressions. 2. Other mouse models. Mouse models of other familial cancer syndromes have sometimes resembled their human counterpart. Both P53 nullizygous mice (357) and mice heterozygous for P53 mutation (298) develop soft tissue sarcomas, osteosarcomas, and lymphomas, all tumors represented in the Li-Fraumeni syndrome. As noted above, tumors from P53 heterozygous mice, like tumors from patients with germline P53 mutation and the Li-Fraumeni syndrome, frequently retain the wild-type allele, suggesting that p53 haploinsufficiency may trigger tumorigenesis in susceptible tissues. The Men1 / genotype in the mouse is sufficient for tumor predisposition in many of the same tissues affected in the human disease, including the pancreatic islets, parathyroids, adrenal (cortex and medulla), and pituitary (153). Similarly, targeted transgenic expression of a MEN2A-associated RET oncogene in murine thyroid C cells was sufficient for the predisposition to MTC (358), and targeting of the MEN2B-associated Met918Thr RET oncogene to the sympathetic nervous system and adrenals was sufficient for the development of ganglioneuromas in transgenic mice (359). Heterozygous or homozygous inactivation of the cas-r in the mouse was sufficient to recapitulate the principal metabolic disturbances of human FHH and NSHPT, respectively (230). Mice nullizygous for VHL died in utero, whereas VHL / mice were phenotypically normal (360), indicating that a satisfactory mouse model for VHL has not yet been developed. Mouse models of tumorigenesis allow the controlled introduction of one or two hits, sometimes making the anticipation, harvest, and molecular genetic analysis of early stages in tumorigenesis possible. For example, in a model for insulinoma islet -cell-specific knockout of Men1, the stepwise evolution of pancreatic islet cell tumors from a polyclonal hyperplastic phase to frank adenoma could be studied in detail (291, 361). 3. Models in other species. In large part because of their central roles in cell growth, oncogenes and tumor suppressor genes have their homologs widely through the evolutionary tree. Important information has been developed, particularly from studies in yeast, roundworms, flies, and fish. However, because hormonal systems are poorly developed and/or poorly understood in these species, little of the information is presently relevant to secretion by hormonal tumors in humans. For the same reasons, mutations in known hormonal signaling genes have not been extensively extended to these species. VI. Tissue Specificity in Hereditary Hormone Excess A. Expressions of tissue specificity in hormone excess syndromes 1. Different genes cause hyperfunction of the same cell type. By virtue of a focus on hormone excess, this article collects syndromes and mutated genes that target limited types of cells and not surprisingly with some overlaps of these targets. Still, the large number of known genes that can target a single tissue type for hormone excess through germline mutation is remarkable (Table 6). This is all the more impressive because additional genes with the same targets remain to be identified. For hereditary tumors, a germline mutation is, by definition, the factor predisposing to tumors, but stepwise changes at the same gene (second hit) and other genes (third hit and beyond) are required for tumor progression. These later hits may even contribute to tissue specificity of the tumor. For example, this seems likely for pheochromocytoma in MEN2, where RET activating mutation is frequently accompanied by biallelic inactivation of VHL (170). Similarly, in mice with biallelic knockout of Men1 (which is on murine chromosome 13), insulinomas showed gene amplification on chromosome 11, but prolactinomas showed gene amplification on chromosome 15 (291). 2. One gene or one codon gives a selective expression in a cell type. The expression details of hyperfunction within a cell type can vary among hormone excess syndromes, predisposed from different mutant genes (Table 6). The most obvious differences in expression are between genes causing hormone excess from monoclonal vs. polyclonal hyperfunction (167). However, even within the monoclonal or polyclonal group, there are gene-dependent differences in tumor penetrance, aggressiveness, and histology. For example, the aggressiveness of adrenocortical tumor can vary from frequent malignancy with hypersecretion of multiple steroids (from P53 mutation), to benign with hypersecretion of corticosteroids (from mutation of PRKAR1A or GNAS mutation), to benign without steroid hypersecretion (from MEN1 mutation). Even within a single gene, particularly RET, the mutated codon may determine the details of its effect, causing three discrete grades of MTC aggressiveness.

28 642 Endocrine Reviews, August 2005, 26(5): Marx and Simonds Hereditary Tumors TABLE 6. Different genes through germline mutation can predispose to hyperfunction in the same cell type a Hyperfunctioning cell type Mutant gene in germline b 3. Effects of a neoplasm on secretion of its hormone(s). Hormone secretion can be disturbed by changing secretory mass and/or by changing the regulation of hormone secretion. Abnormalities can include acquired secretory responses regulated by ectopic receptors (362). Ectopic release of hormones by neoplasms is not covered herein. The effect on cell mass is obvious. Most endocrine tumors have a tissue mass that is more than 20-fold the mass of the normal gland. Even allowing for lower hormone storage per cell, the hormone secretory mass is markedly increased. Increased secretory mass may sometimes elevate the nonsuppressible basal output of hormones (363). Regulation of hormone output by tumors has received limited attention, and even less in the more rare hereditary tumors. In general, hormonal neoplasms retain important aspects of the normal mechanisms for regulating secretion (364, 365). Not surprisingly, lesser degrees of normal regulation may persist with malignant than with benign hyperfunction of hormone-secreting tissues (366). 4. Associated monoclonal neoplasia in hormone nonsecreting tissues. Careful analysis of large families and improved precision of syndrome diagnosis from analysis of gene mutations have led to greater numbers and variety of the tumor types attributable to a syndrome (367, 368). These have added to evidence that the majority of hereditary hormonal syndromes cause also nonhormonal neoplasms; most of these are stromal or mesenchymal tumors (Table 3). In fact, most of the MENs have less prominent expressions in hormonal than in nonhormonal tissues (see Section IV.C). Syndromes and mutant genes with only hormonal neoplasms are the Gene-specific special feature in hyperfunctioning cell Parathyroid cell MEN1 / Frequently recurs after subtotal parathyroidectomy (216). RET */ Hyperparathyroidism with MEN2A, but not with FMTC or MEN2B (50). HRPT2 / Often malignant. Often cystic (76). CASR / Atypical HPT. Polyclonal. Minimal hyperplasia (167). CASR / Very severe HPT in neonate. Likely polyclonal (167). Pancreatic -cell MEN / Typical insulinoma features (216). VHL / Usually hormone nonsecreting. Often cystic (178). SUR1 / Atypical hyperinsulinism. Polyclonal (167). Kir6.2 / Atypical hyperinsulinism (167). GK */ Atypical hyperinsulinism. Maybe later onset (239). GLUD1 */ Atypical hyperinsulinism (167). Somatotrope MEN1 / Nearly all are macroadenoma. Prolactinoma more frequent in MEN1 (60). PRKAR1A / GH cell hyperplasia abuts tumor (368). GNAS */ c Prolactin cosecretion (455). Chromaffin cell VHL / Only from selected mutations (176). Age of onset earlier and catecholamin profile unlike pheochromocytoma in MEN2 (165). RET */ Only from selected mutations. Hyperplasia as precursor in MEN2 not VHL (177). SDHB / Mainly pheochromocytoma (82). SDHC / Mainly paraganglioma (82). SDHD / Mainly paraganglioma. Imprinted gene. Maternal allele silent (82). NF1 / Low penetrance of pheochromocytoma (200). MEN1 / Low penetrance of pheochromocytoma (55). Adrenal corticotrope PRKAR1A / Pigmented, macronodular. High cortisol (187). GNAS */ c Age below 1 yr. Hyperplasia (M. Collins, personal communication). MEN1 / Most frequently hyperplastic and steroid nonsecreting (156). P53 / Mainly in child, low penetrance but often malignant (130). a Details of expression can also derive from selected mutation within one gene, exemplified by varying age at onset of C cell cancer dependent on codon of the RET mutation. b Alleles are:, wild type; *, gain-of-function mutation;, loss-of-function mutation. c GNAS mutation in McCune-Albright syndrome is not germline but postzygotic. exceptions and include all the MEN2 variants (if ganglioneuromas are judged as overlapping with neuroendocrine tumors) and syndromes of hormone excess involving only a single tissue that is hormonal. B. Mechanisms for tissue specificity of hormonal excess syndromes 1. Tissue targeting by a promoter. A promoter serves several tasks in its broad function to regulate the initiation of mrna synthesis. Targeting of its mrna expression to all, several, or only one selected tissue is a major role of a promoter, in combination with promoter availability in chromatin and with gene-interactors and chromatin-interactors in the cell. A series of 39 homeobox-containing or HOX genes help, together with other transcription factors, establish tissue identities during development (369). a. Mutation within a promoter. One explanation for tissue specificity of a tumor is a mutated promoter. Work with transgenic animals has led to many models in which a tumorigenic germline rearrangement (i.e., mutation) is engineered by fusing a tissue-specific promoter so that it drives expression of an oncogenic protein into a localized tissue. The rat insulinoma models, such as RIP-TAG (short for Rat Insulin Promoter-fused to large T AntiGen), have been studied in great detail (370). Similar natural tumorigenic DNA rearrangements have been discovered in somatic genes of man, such as fusion of the PTH promoter with the cyclin D1 open reading frame in parathyroid adenoma. Such a tumor-

29 Marx and Simonds Hereditary Tumors Endocrine Reviews, August 2005, 26(5): TABLE 7. Examples of possible coactivators toward the tissue specificity of hormonal tumors, predisposed by a broadly expressed gene Possible coactivators for tumorigenesis Target cell type Endogenous Exogenous Parathyroid cell Hypocalcemia; defect of vitamin D receptor or of 25OHD 1 -hydroxylase Lithium; calciolytic drugs; neck radiation; diet low in calcium, low in vitamin D, or high in phosphate Lactotrope Estrogen receptor; estrogen Estrogenic or neuroleptic drugs Gastrin cell Achlorhydria; CDK2A inactivation (286) Blockers of H -pump or H 2 histamine receptor Adrenal corticotrope ACTH RU-486 Thyrocyte TSH; RET-PTC Radioactive iodine; neck radiation; iodine deficiency Chromaffin-chemosensory cell Hypoxia High altitude Chromaffin-adrenomedullary cell in MEN2 VHL inactivation (170) Glucagon Evidence for most potential coactivators is speculative and/or weak and derived mainly from sporadic tumors. igenic promoter fusion has not been identified in the human germline. b. Mutation upstream of a promoter. A related explanation for tissue specificity is a mutant protein either upstream or downstream of a tissue specific promoter, with the promoter still conveying tissue selectivity. Thus, selectivity could arise from an upstream mutation that acts upon a component of a transcriptional complex that binds to a tissue-specific promoter. Somatic mutation in many leukemias or lymphomas activates such a transcription factor to act upon genes that are tissue-selective for hematopoetic development (371). c. Mutation downstream of a promoter. A normal tissueselective promoter can drive the expression of a mutated protein at one or further steps downstream. This is applicable for certain hereditary nonhormonal tumors, such as multiple exostoses syndromes (372) and multiple enchondromas (373). This is also relevant for RET and PTEN, whose expressions are high within their tumor target tissues ( ). In like manner, syndromes of polyclonal hormone excess, like other polyclonal syndromes that are not hormonal, typically reflect mutation in the open reading frame of a gene that normally has the same tissue-specific expression. The CASR is most intensely expressed in the parathyroid cell (378). The TSHR is selectively expressed in the thyrocyte (379). Regulation of expression of the LHR is complex and with main expression in steroidogenic tissues (380). It is believed that normal expression of SUR1, Kir6.2, GK, and GLUD1 is each limited mainly to the pancreatic -cell (381, 382); only GLUD1 is expressed in hepatocyte also, where its germline activating mutation in PHHI causes hyperammonemia but not tumor (241). 2. Hypotheses for the tissue specificity of a tumor, predisposed by a broadly expressed gene. Most genes that are tumorigenic in a selected tissue are normally expressed in all or most tissues (for example, RB1, P16, GNAS, PRKAR1A, MEN1, VHL). Several mechanisms have been speculated for the tissue specificity of their hormonal neoplasias. In fact, different mechanisms may apply to different genes. Furthermore, the few examples of tumor tissue specificity that are understood may not be relevant to understanding tissue targeting of most hormone excess syndromes. These hypotheses can also be conceptualized as those with a special feature in the targeted tissue (redundancy hypothesis; coactivator hypothesis) and those with a special feature in the protected tissues (suppressor hypothesis); at the same time, it is evident that each must postulate some other difference between targeted and protected tissue. a. Redundancy hypothesis. The redundancy hypothesis is specific to growth suppressors that cause tumors by loss of gene function. This hypothesis states that a growth suppressor gene has backup processes in most tissues. However, it has inadequate backup in selected tissues, and this makes those tissues specifically susceptible to inactivating mutation in that gene (383). Some tumor suppressor genes belong to families of closely related genes (P53, PRKAR1A, RB1); thus, some of the fully homologous proteins or proteins with homologous domains may serve such a backup role (384). Several other tumor suppressor genes have no homologs (MEN1, VHL). For these genes, nonhomologous proteins must be the hypothetical backups. b. Coactivator hypothesis. The coactivator hypothesis speculates that a tissue-specific coactivator can or must synergize with the mutant gene to cause a tumor. The coactivator could be endogenous or exogenous (Table 7). The concept of the cancer modifier overlaps with that of the endogenous coactivator (385). Xeroderma pigmentosa arises from mutation in any one of seven genes, presumed to be widely expressed (386); because of the requirement for ionizing (i.e., sun-derived) radiation to synergize with the germline mutation in tumor development, the dermis is specifically targeted for tumors (basal cell carcinoma, squamous cell carcinoma, melanoma). Other tumors mainly limited to the exposed facial skin may have a similar contribution from solar radiation; these include angiofibroma in MEN1 or in tuberous sclerosis. The coactivator hypothesis would also accommodate the speculation that diagnostic x-rays about the neck promote the development of parathyroid tumors or C cell tumors in some syndromes with predisposition to either or both (387). It would also accommodate a role for third hit and beyond (see Section V.A.4). Many examples of endogenous or exogenous coactivators can be speculated, particularly by invoking a coactivator role for selected medications (Table 7). Some speculated coactivators are known carcinogens (radioactive iodine for the thyroid;

30 644 Endocrine Reviews, August 2005, 26(5): Marx and Simonds Hereditary Tumors VHL inactivation for adrenal medulla in MEN2); most are only known to be stimulators (directly or indirectly) of proliferation. c. Suppressor hypothesis. In contrast, the suppressor hypothesis states that the second-hit or stepwise abnormality leads to death of the isolated mutated cell in most tissues, and such a second hit is thereby invisible or normal, but cell death is not caused by the mutation in the disease target tissue, where instead a tumor clone will progress. A tissue-specific factor may protect the tumor target tissue from death; this variation incorporates features of the coactivator hypothesis in assigning a tissue-selective role to interacting protein(s) (383, 388). Against this hypothesis, homozygous absence of menin in the liver did not cause hepatocyte dysfunction (388). VII. Syndrome and Gene Classification According to Mutated Process 5 and Pathway A. Overview of downstream pathways and broad endpoints At the cellular level, the two broad endpoints contributing to hormonal excess can be classified as cell accumulation and terminal differentiation. Terminal differentiation here includes the apparatus for synthesis and secretion of hormones. Several alternative subgroups can be used to classify the critical biological pathways for cell accumulation and for terminal differentiation. Selected recent reviews of many of the principal topics are indicated below. Five categories in wide use, although not encompassing all cell functions, are cell birth, cell death, housekeeping, genome stabilization, and secretion. Cell birth includes angiogenesis and the response to hypoxia (389), the cell cycle (345, 390), mitosis (391), and telomerase (392). Cell death (393, 394) includes TNFrelated pathways (395), the caspases (396), ubiquitinylation (397), and the 26S proteasome (398). Housekeeping functions include matrix synthesis, adhesion, chromatin remodeling (399), transcription (400), and translation (401). Genome stabilization includes DNA checkpoints and DNA repair ( ). Hormone secretion includes sensing of extracellular input (405), its downstream signal transduction (209), and hormone synthesis, sorting (406), and exocytosis (407). In focusing upon pathways, this paper extends the traditional metabolic biosynthesis-type approach, which assumes that many pathways are organized as a linear chain. This is illustrated by a similar syndrome of PHHI that can result from mutation at any of three steps (Fig. 2). Some recent data support another paradigm that has not been fully developed. A protein may act on one or several partners resulting in an exponentially expanding downstream network. For example, one expression of this viewpoint is that one transcription factor may regulate a significant fraction of all the expressed genes in a tissue (408). B. Hypoxia/angiogenesis pathways Angiogenesis participates in tissue development, tissue growth, and tissue repair (389). Angiogenesis in most solid 5 Herein the term process implies that incomplete information about the more extended pathway is available (427, 434). malignancies is manifested in part by high concentrations of VEGF and VEGF receptors; this is particularly prominent in benign paraganglioma and pheochromocytoma (84, 179). Because it is so prominent in neoplasms, angiogenesis is under evaluation as a target for development of anticancer drugs. Hypoxia within growing tissues such as tumors is a major stimulus for angiogenesis. Hypoxia is detected by intracrine mechanisms, by the specialized paraganglionic sensory tissues, and by the renal peritubular fibroblasts that synthesize erythropoietin (409). In all tissues, hypoxia stimulates synthesis of HIF. HIF is a dimer of two homologous subunits (HIF-1 and HIF-1 ); HIF belongs to the basic helix-loophelix family of transcription factors. HIFs bind to and activate cis-promoter elements of many genes, including those for VEGF and erythropoietin (410). The HIFs may have inherent tumorigenic properties (411). Of the four known genes (other than those in mitochondrial complex II) that can cause hereditary pheochromocytoma (Tables 1 and 6), VHL alone has a molecular action recognized to overlap by a known mechanism with the hypoxia/angiogenesis pathway. All tumors in VHL have a substantial vascular component. The pvhl protein participates in complexes with ubiquitin ligase activity. Perhaps the most critical substrate for vhl-dependent ubiquitinylation is the -subunit of HIF-1; as a probable result of loss of this ubiquitinylation, HIF-1 accumulates in tumors of VHL (176). HIF is also regulated by hydroxylation on proline and asparagine (412). This process is dependent on 2-oxoglutarate, which can be metabolized to succinate, offering a possible interaction with the mitochondrial sdh enzymes and PGL syndromes (413). Several genes herein are likely to be involved in pathways of oxygen sensing and/or angiogenesis, and their mutation can cause paraganglioma (SDHB, SDHC, SDHD, but not SDHA) or the closely related pheochromocytoma (VHL). The four sdh subunits are part of the mitochondrial complex II of the respiratory chain. Complex II functions in the Krebs cycle to catalyze oxidation of succinate to fumarate. Functioning in reverse, it can catalyze fumarate reductase in anaerobic electron transfer. C. Cell cycle pathways Genes at many steps in cell cycle regulation have been shown to participate in hereditary tumorigenesis mainly through alterations of their regulation and expression. Germline mutation of none causes hereditary hormone excess. However, the cyclin D1 gene through somatic mutation has special relevance. It has been implicated by somatic mutation and by other roles as a tumor promoter in many sporadic malignancies, including mantle cell lymphoma, breast cancer, and parathyroid adenoma. CYCD1 mutation, through 5 fusion to the PTH promoter, is central to monoclonal growth in 4 5% of nonhereditary parathyroid tumors. D. Apoptosis pathways Programmed cell death or apoptosis removes cells in contexts that vary from anabolic to catabolic. Apoptosis was first outlined in Caenorhabditis elegans development, and much of

31 Marx and Simonds Hereditary Tumors Endocrine Reviews, August 2005, 26(5): its molecular machinery is conserved between C. elegans and man (393). This pathway can be initiated by cell surface death receptors in the TNF-R superfamily (395). Another branch of this pathway with Bax and Bcl2 originates with intracellular events, such as DNA damage and signals at the mitochondrial membrane. Both initiators signal to the intracellular cascade of some 15 caspase proteolytic enzymes. Many genes within and related to the apoptosis pathways can contribute to hematological monoclonal neoplasia through somatic mutation (394). Despite this and despite phenotypes from mutation of many apoptosis genes in animal models, no disease from germline mutation of one of these genes has been recognized in man. P53 is a tumor suppressor gene, and its germline inactivation causes of the Li-Fraumeni syndrome (see Section IV.A.5). Its downstream actions include G 1 arrest by stimulating P21, G 2 checkpoint regulation, and apoptosis promotion. P53 directly stimulates transcription of several proapoptotic genes, including BAX, BCL-2 homologs (PUMA and APR), and TNF-R related genes (FAS and DR5) (414, 415). E. Housekeeping processes Housekeeping refers to proteins that serve functions that are stable through the cell cycle. The background concentration of the protein may be low or high, but the concentration fluctuates little. Because the concentrations are steady, their regulation is not prominent, and these proteins are often not described as part of a pathway. At the same time a housekeeping process does not preclude involvement within an otherwise regulated pathway. Some of the functions include cell adhesion, maintenance of extracellular matrix, many enzymes, transcription, and translation. Germline mutation in some has been recognized as the cause of some hereditary neoplasias [EXT1 or EXT2 for multiple exostoses; and SNF5/INI1 for rhabdoid tumors (51)]. MEN1 and HRPT2 were suggested each to function in a different large complex with polymerase II (111, 163). F. Genome stabilization pathways Hereditary defects in homologous repair of doublestranded DNA breaks (i.e., homologous replication repair) result in DNA susceptibility to mutation (402). Typically, such defects predispose to neoplasia of the lymphoid system and skin (ATM for ataxia telangiectasia; NBS1 for Nijmegen breakage syndrome; MRE11 for ataxia telangiectasia-like; BLM for Bloom syndrome), reflecting susceptibility to DNA damage from ionizing radiation. Several of these defects result, rather selectively, in increased susceptibility to breast cancer (BRCA1 or BRCA2 for familial breast and ovarian cancer; ATM heterozygote has mildly increased susceptibility to breast cancer). Werner syndrome, like several hereditary defects, is from a mutation in other or still unknown pathways of genome stability, perhaps linked to homologous replication repair. Werner syndrome (from WRN mutation) expresses increased rates of nonepithelial cancers, including non-mtc (see Section IV.B.4). The defect is in a helicase for DNA or RNA with unknown normal role; BLM encodes another helicase with weak homology to WRN. Fanconi anemia is a homozygous disorder that predisposes to acute myeloid leukemia and squamous cell carcinoma. There is a defect of DNA crosslink repair. Mutation in any one of seven different genes, including BRCA2, gives this phenotype. Defects in DNA mismatch repair are expressed mainly as nonpolyposis colon cancer. Germline and sometimes somatic mutation in any one of six genes has been identified: hmsh2, hmsh3, hmsh6, hmlh1, hpms1, and hpms2. The typical finding concerning DNA in tumor is microsatellite instability, but there is also increased mutation in expressed gene sequences. Three distinct autosomal recessive syndromes are associated with defective nucleotide excision repair: xeroderma pigmentosum (XP), Cockayne syndrome (CS), and the photosensitive form of trichothiodystrophy (TTD) (386). Among these three, only XP has an increased rate of cancers, mainly in skin and including melanoma. G. Signal transduction pathways and processes Signal transduction involves a varying input acting on a sensor and transmission of the status of the input via a transducer to another step that leads to an output. The output may be defined at a nearby molecular step downstream from the input, such as G protein-coupled transmembrane receptor (GPCR)-to-cAMP or at a variable number of steps further removed such as hormone secretion. There is generally signal amplification in the signal transduction pathway. A rapid time course of seconds to hours is usually one criterion for a signal transduction process. But slower phenomena, such as regulation of bone mass through the wnt pathway, may also be incorporated into this concept (416). Signal transduction is a broad concept. For example, some components of a signal transduction process may be all or partly extracellular. On the other hand, some other signal transduction pathways are entirely within the nucleus (400). The signal transduction paradigm can be applied to many processes and pathways and need not imply regulation of exocytosis of a hormone. The main utility of the signal transduction concept in this paper is to collect genes and molecules that can be examined for possible contribution to hormone secretion. 1. Syndromes of polyclonal hypersecretion. Three genes (CASR, TSHR, and LHR), each of whose germline mutation can cause a polyclonal syndrome of hormonal excess, encode serpentine, seven-passing, GPCR (Fig. 3). Each of these GPCRs is a plasma membrane sensor for a ligand in blood, and each controls a hormone secretory response to its circulating ligand, responding on a time-frame of seconds. The TSHR and LHR transduce mainly via Gs; the cas-r transducer is presumed to be a different G protein, and this might be Gq (45). The hormones whose downstream secretion is regulated by these three receptors are highly different: a polypeptide (PTH), iodothyronines, and sex steroids. For each of these three serpentine receptors, the final steps of the mutated pathway to hormone secretion remain poorly understood, but major overlaps among the three pathways seem likely (405, 407).

32 646 Endocrine Reviews, August 2005, 26(5): Marx and Simonds Hereditary Tumors FIG. 3. Interactions of genes mutated in cell cycle regulation, as analyzed with mouse models. Color-coding indicates cyclins (yellow), CDK (green), and CDK inhibitors (magenta). Mitogenic signals, particularly those acting through the p21 ras 3ERK pathway, stimulate transcription of cyclin D isoforms, resulting in increased complex formation between CDK4 and CDK6 and cyclin D. The retinoblastoma gene product, prb, in its unphosphorylated state, normally binds to and sequesters the E2F family of transcription factors. Successive phosphorylation of prb by CDK4 and CDK6 (bound to cyclin D) and CDK2 (bound to cyclin E) inhibits its ability to bind and sequester E2F. Upon its release from prb, E2F becomes transcriptionally active and switches on the transcription of multiple genes important for DNA synthesis and progression into the S phase, including cyclins E and A. Members of both the INK4 and Cip/Kip families of CDK inhibitors inhibit the function of cyclin D/ CDK4/6 complexes, whereas members of the Cip/Kip family also inhibit cyclin E/CDK2 and cyclin A/CDK2 complexes. The products of P53 and PTEN can strongly induce the expression of certain CDK inhibitors as shown. Loss of P53 function in the Li-Fraumeni syndrome and of PTEN function in the Cowden syndrome result in decreased expression of the CDK inhibitors p21/waf1 and p27/kip1, respectively, as shown. The CDK inhibitors also function in other phases of the cell cycle not shown here. Depending on the state of the cell cycle and/or phosphorylation, many of the cell cycle regulatory proteins shown can be found in either the cytoplasm or the cell nucleus. Proteins or genes whose loss of function in humans or mice predisposes to monoclonal neoplasia are labeled in italics (see text). The mechanisms whereby heterozygous TSHR gain-offunction causes hyperthyroidism are probably many. camp activates many steps in thyroid function including endocytosis of thyroglobulin and expression of the sodium/iodine cotransporter. Important control of camp over thyrocyte proliferation is likely but not established (417). Considering the large number of additional GPCRs, including some that regulate secretion of hormones, it is likely that analogous mutations remain to be discovered in other GPCRs. This concept was tested in preliminary ways without finding mutation in the GHRH-R or the ACTH-R (36, 418). Any one of four other genes (GK, GLUD1, SUR1, Kir6.2) can be mutated in the germline to cause PHHI and a polyclonal pancreatic -cell hyperfunction. In the pancreatic -cell, the glucose-to-insulin signal-transduction pathway begins with glucose in blood (Fig. 3). Cytoplasmic glucose is converted by GK, the type IV hexokinase (GK mutation is one cause of PHHI), to glucose-6-phosphate. GK is considered to be the glucose sensor of the -cell (254). Glucose-6-phosphate allows increased oxidation of carbohydrate. GlnDH (GLUD1 mutation is another cause of PHHI) in the mitochondria oxidizes glucose-6-phosphate to -ketoglutarate, simultaneously gener-