SEARCHING FOR THE HEREDITARY CAUSES OF RENAL-CELL CARCINOMA

Transcription

1 SEARCHING FOR THE HEREDITARY CAUSES OF RENAL-CELL CARCINOMA *Christian. avlovich and Laura S. Schmidt Families with hereditary predispositions to cancer continue to provide a unique opportunity for the identification and characterization of genes involved in carcinogenesis. A surprising number of genetic syndromes predispose to the development of renal-cell carcinoma, and already genes associated with five of these syndromes have been identified VHL,, FH, BHD and HRT2. These very different genes and the biochemical pathways in which they participate raise interesting questions about the development of renal cancers and could lead to new therapeutic approaches in the near future. So, what is known about hereditary renal cancer at present? KREBS CYCLE Also known as tricarboxylic-acid cycle. A series of enzymatic reactions that break down pyruvate to carbon dioxide and hydrogen atoms, which are in turn transferred to specific coenzymes for the oxidative generation of AT in the mitochondria. GENODERMATOSIS An inherited syndrome involving a dermatological phenotype and possibly other phenotypes. * Johns Hopkins Bayview Medical Center, Brady Urological Institute, A-345, 4940 Eastern Ave., Baltimore, Maryland 21224, USA. Basic Research rogram, SAIC-Frederick, Inc., National Cancer Institute at Frederick, Bldg 560, Rm , Frederick, Maryland 21702, USA. Correspondence to C.. or L.S.S. s: cpavlov2@jhmi.edu; schmidt@ncifcrf.gov doi: /nrc1364 Renal-cell carcinoma (RCC) affects approximately 150,000 people worldwide each year, causing close to 78,000 deaths annually, and its incidence seems to be rising 1,2.RCC is not a single entity, but rather comprises the class of tumours of renal epithelial origin. Extensive histological and molecular evaluation has resulted in the development of a consensus classification of different RCC subtypes (TABLE 1) 3.Although most cases of RCC seem to occur sporadically, an inherited predisposition to renal cancer accounts for 1 4% of cases and could involve the same genes that cause sporadic renal cancer. Over the past two decades, studies of families with inherited RCC have laid the groundwork for the identification of seven hereditary renal cancer syndromes, and the predisposing genes for five of these have been identified (TABLE 2).The surprisingly diverse nature of these genes implicates various mechanisms and biological pathways in RCC tumorigenesis. The year 2003 marked the tenth anniversary of the discovery of the von Hippel Lindau (VHL) tumoursuppressor gene 4, the first gene identified for hereditary RCC that is now known to be involved in the most cases of sporadic RCC. The VHL gene product is involved in the regulation of numerous pathways leading to extracellular-matrix assembly, cell-cycle regulation and, most importantly for tumorigenesis, oxygen sensing. Four years after the discovery of VHL, interest in a gene known for nearly two decades to have oncogenic potential was rekindled when activating renal-cancer-causing mutations in patients with hereditary papillary renal carcinoma (HRC) were identified in the protooncogene 5.Recently, the gene that encodes the KREBS CYCLE enzyme fumarate hydratase (FH) was found mutated in renal tumours from patients with a rare GENODERMATOSIS termed hereditary leiomyomatosis and renal-cell cancer (HLRCC) 6.Finally, new renal-cancer-predisposing genes were identified through linkage analysis in families with another genodermatosis, the Birt Hogg Dubé syndrome (BHD) 7, and in families with hyperparathyroidism-jaw tumour syndrome (HT-JT) 8. These predisposing genes BHD and HRT2, respectively are suspected to act as tumour suppressors, although their biological functions are as unknown. Several renal-cancer-associated syndromes have been identified for which no predisposing gene has been found. Several families carry a balanced chromosome-3 translocation that predisposes family members to clearcell RCC in the absence of germline VHL inactivation Other families have members affected with clear-cell renal carcinomas, but no detectable VHL inactivation or germline chromosome-3 translocations. Finally, familial papillary thyroid carcinoma (FTC), which predisposes patients to thyroid cancer and nodular thyroid disease, can also predispose to papillary RCC and ONCOCYTOMA 12. NATURE REVIEWS CANCER VOLUME 4 MAY

2 Summary A predisposition to renal cancer has been identified in several autosomal-dominant inherited cancer syndromes. von Hippel Lindau (VHL) disease, associated with conventional (clear-cell) renal-cell carcinomas and multi-organ neoplasia, is caused by germline mutations in the VHL tumour-suppressor gene and loss of the wild-type VHL allele. atients with hereditary papillary renal carcinoma (HRC) harbour germlineactivating mutations in the proto-oncogene, which can cause renal cancers with papillary type-1 histology. apillary type-2 renal carcinomas and cutaneous and uterine smooth-muscle tumours are associated with the syndrome of hereditary leiomyomatosis and renal-cell cancer (HLRCC), which is caused by germline loss-of-function mutations in the fumarate-hydratase (FH) gene. The Birt Hogg Dubé syndrome (BHD) predisposes to cutaneous nodules (benign tumours of the hair follicle), spontaneous pneumothorax and an increased risk for renal cancers of various histological types, such as chromophobe renal-cell carcinoma and oncocytic hybrid renal tumour. BHD is caused by germline mutations in a newly discovered tumour-suppressor gene, BHD. Hyperparathyroidism-jaw tumour syndrome (HT-JT) is associated with parathyroid adenomas, fibro-osseous tumours of the jaw, and unusual renal tumours containing a mixture of epithelial and stromal elements. This syndrome is caused by germline mutations in HRT2. The identification of non-vhl families affected with clear-cell renal carcinomas, termed familial clear-cell renal carcinoma (FCRC), indicates that additional renalcancer-predisposing genes remain to be identified. Diagnosis and appropriate treatment of these hereditary renal-cancer-associated syndromes relies on an understanding of their clinical spectrum, accurate histological evaluation of renal tumours from patients and on genetic testing for predisposing genes. ONCOCYTOMA Large tumour cells with poorly defined borders, granular eosinophilic cytoplasm and large, basophilic nuclei. This review will focus on the syndromes that are associated with hereditary renal cancer, the attempts to understand the molecular biology of the genes already discovered, and on the search for new predisposing genes. Two forms of renal tumour that are associated with hereditary syndromes will not be covered angiomyolipoma associated with tuberous sclerosis, and hereditary Wilms tumour as both are not strictly of renal epithelial origin and both have heritable genetics that have recently been reviewed elsewhere Hereditary syndromes with known genes VHL syndrome. VHL disease, named after the physicians who first described the hallmark retinal angiomas and central nervous system (CNS) haemangioblastomas, is an autosomal-dominant, inherited multisystem disorder that is characterized by solid vascular tumours of the kidney, CNS, retina, adrenal gland and endolymphatic sac, and by vascular/cystic lesions of the kidney, pancreas, epididymis and broad ligament 16. VHL occurs at a prevalence of about 1/35,000 and VHLassociated tumours with relatively high ENETRANCE (80 90%) develop in the second to fourth decades of life. The renal tumours are almost exclusively clear-cell renal carcinomas or cystic variants that are often, but not always, multifocal and bilateral, and usually acquire metastatic potential when they reach more than 3 7 cm in diameter (TABLE 1). VHL disease is caused by germline mutations in the VHL tumoursuppressor gene 4,17,18 accompanied by inactivation of the wild-type copy of the VHL gene in a susceptible cell through LOSS OF HETEROZYGOSITY (LOH), promoter HYERHYLATION or somatic mutation, according to the KNUDSON TWO-HIT TUMOUR-SURESSOR MODEL In fact, the two-hit event is documented very early in microscopic pre-neoplastic renal lesions and cysts 22,23.Over half of all sporadic conventional renal carcinomas carry biallelic inactivation of both VHL alleles Since the cloning of the VHL gene in 1993, an enormous number of experimental studies have been undertaken to unravel the pathway by which VHL inactivation leads to cancer (for reviews, see REFS 27 29). The frequent overexpression of vascular endothelial growth factor (VEGF) and erythropoietin (EO) in VHL-associated tumours, both of which are encoded by hypoxia-inducible genes, offered clues to a potential role for VHL in regulating genes that are controlled by cellular oxygen tension (for reviews, see REFS 30,31). Subsequently, VHL was found to regulate expression of hypoxia-inducible factor (HIF) 32,a heterodimeric protein made up of -subunits (HIF-1, HIF-2 or HIF-3) and HIF-1 33.Under normoxic conditions, the -subunit of HIF is hydroxylated at two proline ENETRANCE The frequency with which individuals who carry a given gene mutation will show the manifestations associated with the gene syndrome. If penetrance of a disease allele is 100%, then all individuals carrying that allele will express the associated phenotype. LOSS OF HETEROZYGOSITY (LOH). In cells that carry a mutated allele of a tumoursuppressor gene, the gene becomes fully inactivated when the cell loses a large part of the chromosome carrying the wild-type allele. Regions with a high frequency of LOH are believed to harbour tumoursuppressor genes. Table 1 Classification schema for renal epithelial tumours Histological type Cell of origin Behaviour Genes implicated * Chromosomal abnormalities Conventional (clear-cell) roximal renal tubule Malignant VHL, BHD -3p, +5q, -Y, -8p, -9p, -14q; renal-cell carcinoma t(3;5)(p;q) apillary renal-cell roximal renal tubule Malignant, FH, HRT2 +7,+17, -Y, +12, +16, +20; carcinoma t(x;1)(p11.2;q21.2), t(x;17)(p11.2;q25.3) Chromophobe renal Intercalated cell of Rarely BHD -1, -2, -6, -10, -13, -17, -21 carcinoma renal collecting duct malignant Oncocytoma Intercalated cell of Benign BHD -1, -Y; t(5;11)(q35;q13), renal collecting duct t(9;11)(p23;q13) Collecting-duct Renal collecting duct Aggressively FH -1q32, -6p, -8p, -21q carcinoma malignant * Genes potentially involved in sporadic neoplasms of each particular type, which have been identified by sequence abnormalities found in cases of hereditary renal tumours of similar histology. Rarely metastasize if less than at least 3 cm in diameter; if bigger than this, tumours have an increase in metastatic potential. Tumours smaller than this are occasionally classified as tumours of low malignant potential or as adenomas. BHD, Birt Hogg Dubé (encoding folliculin); FH, fumarate hydratase; HRT2, hyperparathyroidism 2; VHL, von Hippel Lindau. 382 MAY 2004 VOLUME 4

3 Table 2 Heritable syndromes associated with renal-cell neoplasia Syndrome Causative gene, Renal manifestations Other manifestations location Von Hippel Lindau (VHL) VHL, 3p25 Clear-cell RCC: solid and/or cystic, Retinal and CNS haemangioblastomas; multiple and bilateral pheochromocytomas; pancreatic cysts and neuroendocrine tumours; endolymphatic-sac tumours; epididymal and broad-ligament cystadenomas Hereditary papillary renal, 7q31 apillary RCC type 1: solid, multiple None carcinoma (HRC) and bilateral Hereditary leiomyomatosis FH, 1q42 43 apillary RCC type 2, collecting-duct Uterine leiomyomas and leiomyosarcomas; renal-cell cancer (HLRCC) carcinoma: solitary, aggressive cutaneous nodules (leiomyomas) Birt Hogg Dubé (BHD) BHD, 17p11.2 Hybrid oncocytic RCC, chromophobe Cutaneous papules (fibrofolliculomas); lung RCC, clear-cell RCC, oncocytoma: cysts, spontaneous pneumothoraces, multiple, bilateral possibly colon polyps Hyperparathyroidism-jaw HRT2, 1q25 32 Mixed epithelial and stromal tumours, arathyroid tumours, fibro-osseous tumour (H-JT) papillary RCC: cysts mandibular and maxillary tumours Constitutional chromosome-3 Unknown gene, Clear-cell RCC: multiple, bilateral None translocation possibly VHL Familial papillary thyroid cancer Unknown gene, apillary RCC, oncocytoma apillary thyroid cancer, nodular thyroid (FTC) 1q21 disease CNS, central nervous system; FH, fumarate hydratase; HRT2, hyperparathyroidism 2; RCC, renal-cell carcinoma. HYERHYLATION Methylation of a CpG island in a promoter of a gene usually prevents expression of the gene and can be used to regulate gene expression in a tissue-specific manner. Tumour-suppressor genes might be inactivated by mutation or hypermethylation of promoter regions. KNUDSON TWO-HIT HYOTHESIS In 1971, Alfred Knudson proposed that two successive genetic hits are required to turn a normal cell into a tumour cell and that, in familial cancers, one hit was inherited. HEOCHROMOCYTOMA A neuroendocrine tumour that typically arises in the adrenal medulla. These tumours can be benign or malignant. Symptoms often relate to the ability of these tumours to secrete catecholamines. residues by an oxygen-dependent mechanism involving one of several prolyl hydroxylases 34. Crystallographic studies have shown that VHL, through its -domain, binds to elongin C and forms a complex with elongin B 35 and CUL2 (REF. 36) to produce the VHL SCF-like E3 ubiquitin ligase complex. This complex, through VHL, recognizes and binds to HIF- after prolyl hydroxylation and polyubiquitylates HIF- 41, targeting it for destruction by the proteasome 42.During hypoxic conditions, this oxygendependent prolyl hydroxylation can not take place, and HIF- accumulates, moves into the nucleus, dimerizes with HIF-1 and activates expression of hypoxiainducible genes (FIG. 1). A second hypoxia-sensing region of HIF-, the carboxy-terminal transactivation domain (CTAD), binds transcriptional co-activators p300 and CREB-binding protein (CB) to activate transcription of HIF-regulated genes under hypoxic conditions. Hydroxylation of a crucial asparaginyl residue in CTAD by factor inhibiting HIF-1 (FIH-1) during normoxia negatively regulates the function of the HIF- transactivation domain by preventing recruitment of p300/cb 43,44. Experiments in nude mice have confirmed that downregulation of HIF- is required for tumour suppression by VHL 45. VHL / RCC cell lines produce tumours in nude mice, but re-expression of wild-type VHL prevents tumorigenesis, an effect that is, in turn, reversed by expression of mutant HIF-2 46,47.Therefore, the absence of functional VHL as occurs in renal cells with VHL inactivation that have also lost their wild-type VHL allele prevents degradation of HIF- by the proteasome and mimics the cellular response to hypoxia. The hypoxic response, as a result of dysregulation of HIF- subunits, results in transcriptional activation of hypoxia-inducible genes (FIG. 1).These genes encode growth and angiogenic factors such as VEGF, EO and platelet-derived growth factor- that enhance neovascularization of these proliferating renal tumours In addition, transforming growth factor- (TGF-), another HIF target gene, might function in the development of renal tumours, as TGF- and its receptor, epidermal growth factor receptor, are commonly overexpressed in renal carcinoma 53,54.Genes encoding enzymes that are involved in glucose uptake and metabolism (glucose transporter 1 and phosphoglycerate kinase), ph regulation (carbonic anhydrase 9) and tissue-matrix metabolism (matrix metalloproteinases) are also transcriptionally activated by HIF. Additional functions of VHL are rapidly emerging. VHL is clearly involved in assembly of the extracellular fibronectin matrix 55 and has been implicated as a regulator of epithelial-cell differentiation and cell-cycle exit 56 59,perhaps related to its ability to downregulate cyclin D1 (REFS 59,60). VHL also downregulates the chemokine receptor CXCR4, which is involved in organ-specific metastasis and is overexpressed in VHLdeficient renal cancers from individuals with poor tumour-specific survival 61.However, how these functions relate to the development of VHL-associated tumours remains to be established. Interesting genotype phenotype correlations are emerging for VHL disease that relate to the development of RCC. A group of VHL mutations termed type 1, comprising mostly deletions and premature-termination mutations that cause total loss of VHL function, predispose to the entire spectrum of VHL-syndrome manifestations except HEOCHROMOCYTOMAS.By contrast, type 2 mutations, which are mostly missense changes that reduce VHL activity, predispose to the entire VHL spectrum, including pheochromocytomas with or without RCC, called type 2B and type 2A, respectively 62,63. Studies that dissect the molecular consequences of these mutation types on HIF binding 64 have revealed that type-1 and type-2b mutations, which predispose to RCC, show complete loss of HIF-1 ubiquitylation and NATURE REVIEWS CANCER VOLUME 4 MAY

4 OH N HIF-1 OH Elongin B VHL HIF-1 Degradation RBX1 Hypoxia CRE/LOX RECOMBINATION A method in which the Cre recombinase enzyme catalyses recombination between lox sequences. If the lox sequences are arranged as a direct repeat, recombination will delete the DNA between the sites. rolyl and asparaginyl hydroxylases CUL2 Elongin C Ubiquitylation 26S roteasome Fe Elongin B VHL HIF-1 RBX1 VHL inactivation CUL2 Elongin C E3 ubiquitin ligase complex Cytoplasm HIF-1 HRE HIF-1 N HIF-1 HIF-1 p300/cb OH p300/cb Nucleus Transcription of HIF target genes Figure 1 Dysregulation of HIF-1 by VHL inactivation leads to clear-cell renal tumours in patients with von Hippel Lindau disease. Under normoxic conditions, hypoxia-inducible factor-1 (HIF-1) is hydroxylated (-OH) on two conserved proline residues (for simplicity, only one is shown) by a family of prolyl hydroxylases at its oxygen-dependent degradation domain. This hydroxylation provides a substrate-recognition site for the von Hippel Lindau (VHL) E3 ubiquitin ligase complex, which contains elongins C and B, cullin-2 (CUL2) and RBX1. olyubiquitylation of HIF1- by the VHL complex leads to its proteasomal degradation by the 26S proteasome. HIF1- is also hydroxylated at an asparagine residue in its carboxy-terminal transactivation domain by FIH-1, an asparaginyl hydroxylase. This blocks binding of the transcriptional coactivators CREB-binding protein (CB) and p300 to HIF-1, thereby inhibiting transcription of HIF target genes. Hypoxic conditions block both types of hydroxylation, allowing HIF-1 subunits to accumulate and activate transcription of hypoxia-responsive genes. VHL inactivation as occurs in renal cells from patients with a germline VHL mutation and loss of the wild-type allele mimics the hypoxic response by preventing degradation of HIF-1 subunits. Loss of VHL function causes accumulation of HIF-1 subunits in the cytoplasm and their translocation to the nucleus. HIF-1 dimerizes with HIF-1 and is coactivated by CB/p300. HIF/ binds to hypoxia response elements (HRE) in gene promotors, thereby activating transcription of genes upregulated in clear-cell renal tumours, including vascular endothelial growth factor, erythropoietin and platelet-derived growth factor-. (HIF-1 is shown, but HIF-2 is also recognized as a VHL substrate.) regulation, whereas type-2a mutations result in an incomplete defect in HIF regulation. However, type-2a mutations have been shown to disrupt binding of VHL to microtubules and abrogate the associated microtubule-stabilizing function of VHL, implicating defective cytoskeleton organization in this VHL phenotype 65.An interesting third VHL-syndrome subclass (type 2C) predisposes almost exclusively to pheochromocytomas. Type-2C mutations produce VHL that regulates HIF but is defective in fibronectin assembly, indicating a possible link between fibronectin-matrix assembly and pheochromocytoma development 66.Another class of VHL point mutations inactivates VHL function by disrupting proper protein folding mediated by the chaperonin TriC/CCT 67.A recent study of the VHL locus in 55 affected families has demonstrated that families with partial germline VHL deletions are more often affected with RCC than families with complete VHL deletion 68. The development of animal models in which the Vhl gene is deleted should facilitate our understanding of the biochemical consequence of VHL inactivation. Homozygous inactivation of Vhl is embryonically lethal in mice 69 ;however, two mouse models have recently been described in which the Vhl gene was conditionally inactivated using CRE/LOX RECOMBINATION. In the first model, Cre-mediated Vhl inactivation was targeted to the liver and resulted in hepatic haemangiomas 70.In the second model, Vhl was inactivated mosaically in several organs by Cre recombination under a -actin promoter; this produced a highly vascular phenotype, including hepatic haemangiomas, abnormal blood vessels and angiogenesis in several organs, and defective spermatogenesis 71.Interestingly, renal tumours failed to develop in either model, indicating that additional genetic effectors or modifier genes might be necessary for expression of the renal cancer phenotype. The availability of a mouse model in which Vhl is inactivated in the proximal tubule of the kidney will be useful for understanding the renal phenotype of VHL disease. HRC. Families with HRC, which is inherited in an autosomal-dominant pattern, develop multifocal, bilateral papillary RCC with a papillary type-1 histology (TABLE 2). An early report describing ten HRC families noted a late age of onset and a male/female ratio of 2:1 among affected members 72.Metastasis is less frequent, and age-dependent penetrance in mutation carriers seems to be reduced relative to penetrance in VHL syndrome. A genome-wide scan in three families with HRC localized the disease locus to chromosome 7q31, and activating germline mutations were subsequently identified in the tyrosine kinase domain of the proto-oncogene 5, The proto-oncogene encodes a receptor tyrosine kinase that is activated by hepatocyte growth factor (HGF) 76. HGF signalling is important for the EITHELIAL MESENCHYMAL TRANSITION, cell proliferation, branching morphogenesis, differentiation and regulation of cell migration in many tissues, and integration of these pathways causes invasive growth. hosphorylation of crucial tyrosines in the carboxyl terminus of generates a docking site for second messengers (FIG. 2), which activate several signalling pathways involving RAS, phosphatidylinositol 3-kinase, STATs and phospholipase Cγ (for reviews, see REFS 77 79). is overexpressed in many cancers 80,but had not previously been found to be mutated in human cancer. Most of the HRC-associated germline mutations lie within the activation loop or in the AT-binding pocket, and cause ligand-independent activation 81.Several of the HRC-associated mutations lie in codons that are homologous to sites of disease-causing mutations in other receptor tyrosine kinases, indicating that these residues are required for the function of receptor tyrosine kinases 74, MAY 2004 VOLUME 4

5 EITHELIAL MESENCHYMAL TRANSITION Conversion from an epithelial to a mesenchymal phenotype, which is a normal component of embryonic development. In carcinomas, this transformation results in altered cell morphology, the expression of mesenchymal proteins and increased invasiveness. MOSAICISM ostzygotic mutations resulting in some, but not all, of a patient s tissues being affected by gene mutation. atients with mosaicism represent de novo cases of a disease within the family. The patient might be asymptomatic or have less severe disease than offspring and might test negative for a germline mutation, making them difficult to diagnose. NONSENSE MUTATION A sequence alteration in the DNA that changes a codon specific for one amino acid to a chain termination codon, that is, TAA, TAG or TGA. Termination codons produce premature truncated proteins that are likely to abolish protein function. CUTANEOUS LEIOMYOMAS Benign smooth-muscle tumours of the skin presenting as firm, skin-coloured papules and nodules. UTERINE LEIOMYOMAS Also known as a fibroids, benign smooth-muscle tumours of the uterus, the most common gynaecological tumours in women of reproductive age. Fibroids can interfere with childbearing. FUMARASE DEFICIENCY An autosomal-recessive disorder in which biallelic FH mutations cause gross developmental delay and death in the first decade. To understand the mechanism of activation by these mutations, molecular modelling studies of the kinase domain were performed using insulin receptor kinase as a model 74,85.In its inactive form, the kinase domain assumes a bi-lobal configuration, with a selfinhibitory activation loop blocking access of AT to its binding pocket. It is predicted that all of the missense mutations found in the germline of HRC patients either destabilize the self-inhibited inactive form of by displacing the activation loop from the AT-binding pocket, or stabilize the active form, thereby allowing AT to access the binding pocket and activate the kinase. However, addition of HGF to cells expressing mutant further activated the kinase 86,indicating that the need for HGF stimulation for full activation of was lowered, but not completely eliminated, by these missense mutations (FIG. 2).Although two sequential tyrosine phosphorylation events in the catalytic domain are required for wild-type activation, HRCassociated mutants only required phosphorylation of one of the two tyrosines. So, the threshold that is necessary for activation of mutant is lowered as the second phosphorylation event is no longer required 87. Duplication of the chromosome 7 which carries the mutant allele within the patient s renal cells (FIG. 2) provides the second activating event and primes the cell to develop a papillary renal tumour 88,89.It is possible that the additional cytogenetic changes documented in many of these tumours are required for progression from a small papillary adenoma to papillary renal-cell carcinoma (TABLE 1). Met-knockout mice develop an embryonic-lethal phenotype presenting liver, placenta, muscle and nerve defects A mutant mouse model has been described in which one of the HRC-associated mutations was introduced as a transgene; however, these mice develop mammary carcinoma, not renal cancer 93, a phenotype that is also observed in transgenic animals expressing a constitutively activated form of known as TR- 94.Targeted overexpression of HRC-associated mutations in the kidney will possibly produce a mouse model of papillary RCC. HLRCC. Individuals with the autosomal-dominant syndrome HLRCC have an increased risk for papillary RCCs of type-2 morphology, which is an aggressive cancer less commonly encountered than papillary RCC type 1 (REFS 95,96; TABLE 2). apillary RCC type 1 is characterized by cells with scant cytoplasm that are arranged in single layers on papillary cores and often contain foamy macrophages, whereas papillary RCC type 2 is characterized by larger cells with eosinophilic cytoplasm and pseudostratified nuclei 97.Type-2 papillary RCC also differs cytogenetically from type-1 RCC and confers a worse prognosis, as shown in a multivariate analysis that included tumour grade and stage 98,99.The renal tumours in patients with HLRCC are usually found as solitary and unilateral tumours; this is in contrast to those in VHL syndrome, HRC and BHD, which are often multiple and bilateral. In addition, at least two cases of aggressive renal collecting-duct carcinoma have also been described in association with HLRCC 99,100.Lack of multifocal disease in the kidney and the presence of segmental distribution of skin lesions indicate the possibility of MOSAICISM in some affected individuals 99. henotypic features vary among families and among members within families who carry the same germline mutation. In addition, some families develop the cutaneous and uterine features and only rarely renal cancer, a syndrome called multiple cutaneous and uterine leiomyomatosis 101. mutations were not identified in papillary RCC from patients with HLRCC; linkage analysis performed by two independent groups mapped the predisposing gene to chromosome 1q42 44 (REFS 95,101).Subsequently and in collaboration, these groups identified germline HLRCC-associated mutations in FH,a mitochondrial Krebs-cycle enzyme that converts fumarate to malate 6 (FIG. 3). The spectrum of mutations includes missense, insertion/deletion and NONSENSE MUTATIONS that are predicted to truncate the protein, or substitute or delete highly conserved amino acids, along with several wholegene deletions. FH activity evaluated in lymphoblastoid cell lines from patients with HLRCC shows a 20 50% reduction compared with unaffected individuals, and when FH levels were measured in CUTANEOUS LEIOMYOMAS, no activity was observed relative to normal skin samples. So, the consequence of mutations in FH is a severe reduction in enzyme activity. LOH on chromosome 1q42 as well as several acquired somatic mutations have been observed in papillary RCC, and LOH was detected in UTERINE LEIOMYOMAS from patients with HLRCC. These experimental observations clearly support a role for FH as a tumour-suppressor gene. However, unlike VHL,few FH mutations (<2%) have been identified in sporadic-tumours including RCCs, uterine and cutaneous leiomyomas, and sporadic prostate and breast tumours FH mutations are found throughout the entire gene, with no particular genotype phenotype correlations for HLRCC being identified so far. Nearly 40 different mutations have been identified in FH that predispose to cutaneous and uterine leiomyomas and renal cancer 6,99,100.Several of the mutations occur in many families, which could reflect a founder effect; notably, the Arg190His mutation, which is the most frequent mutation (33%) in a North American family study, and the Arg58X and Asn64Thr mutations in studies by the European-based Multiple Leiomyoma Consortium. Arg190His and Lys187Arg mutations were detected in both North American and European families with HLRCC. Types of mutations in patients with HLRCC are similar to those seen in patients with recessive FUMARASE DEFICIENCY, except that the latter tend to be located in the 3 end of the gene. More truncating and whole gene deletion mutations are seen in HLRCC than in fumarase deficiency 105. The overall risk for renal tumour development is unclear and the mechanism of FH-mutation-driven tumorigenesis is unknown at present. One can speculate that defects in the Krebs cycle might lead to blockage and feedback effects on oxidative metabolism and, therefore, the cell cycle. Energy-independent apoptosis is mediated NATURE REVIEWS CANCER VOLUME 4 MAY

6 a Normal renal cell HGF HGF HGF Dimerization Transphosphorylation of catalytic domain Y1234 Y1235 Y1234 Y1235 Docking-site autophosphorylation and secondmessenger binding GAB1 Y1349 Y1356 GRB2 Auto-inhibited b HRC cell with mutation Cell polarity Actin cytoskeleton Motility roliferation Cell-cycle progression Cell-junction formation Migration Invasion Survival HGF HGF HGF Increased activation Y1235 Lower activation threshold Transphosphorylation Y1235 only Y1235 Docking-site autophosphorylation and secondmessenger binding GAB1 GRB2 Y1349 Y1356 Kinase-activated Cell polarity Actin cytoskeleton Motility roliferation Cell-cycle progression Cell-junction formation Migration Invasion Survival Chromosomes Nucleus Duplication of Chr 7 carrying mutant Additional trisomies of Chr 16, Chr 17 and Chr 20 Figure 2 Activating missense mutations in lead to papillary renal carcinoma. a In normal cells, hepatocyte growth factor (HGF) binds the receptor to induce dimerization and release auto-inhibition by the carboxyl terminus. This permits transphosphorylation of catalytic tyrosine (Tyr)1234 and Tyr1235. Subsequent phosphorylation of the multisubstrate docking sites Tyr1349 and Tyr1356 promotes binding of second-messenger molecules, such as GRB2, GAB1, phosphatidylinositol 3-kinase (not shown), and downstream signalling leading to morphogenic, motogenic and mitogenic programmes. b Renal cells from patients with hereditary papillary renal carcinoma (HRC) can harbour germline mutations (star) in the tyrosine kinase domain of. These mutations are predicted to release the auto-inhibition by the carboxyl terminus, allowing the receptor to transition to the active kinase form in the absence of ligand stimulation 85. However, addition of HGF fully activates mutant kinase by stimulation of transphosphorylation of Tyr1235 only 87. Signals for proliferation, invasion and survival occur after docking-site phosphorylation and second-messenger binding. Additional steps such as duplication of mutant -bearing chromosome 7 and trisomy of chromosomes 16, 17 and 20 might be necessary for the development of these late-onset papillary renal carcinomas. 386 MAY 2004 VOLUME 4

7 by oxygen free radicals, and impairment of mitochondrial function leads to severe energy deficits and the generation of large amounts of oxygen free radicals, which cause hypoxia (FIG. 3). Subsequent upregulation of HIF- 1 and transcriptional activation of hypoxia-inducible genes, reminiscent of VHL inactivation, could explain the development of papillary type-2 RCC in response to FH inactivation 106.Recently, germline mutations in succinate dehydrogenase B, another enzyme in the Krebs cycle, were found in patients with hereditary paraganglioma syndrome who developed early onset renal cancer 107, further supporting the idea that impairment of mitochondrial function creates a hypoxic environment that can promote RCC development. Mitochondrion Nucleus Fumarate FH Malate O 2 AT Hypoxia p300/cb HIF-1 stabilization HIF-1 HIF-1 FIBROFOLLICULOMAS Benign hamartomas of the hair follicle, characterized by anastomozing (branching) strands of proliferating epithelial cells extending from a central hair follicle encapsulated by loose, mucin-rich connectivetissue stroma. SONTANEOUS NEUMOTHORAX A sudden rupture of lung tissue, resulting in air escaping from the lung into the pleural cavity. CHROMOHOBE RCC Tumour with well-defined cell borders, fluffy eosinophillic cytoplasm, pyknotic nuclei and perinuclear halos. ONCOCYTIC HYBRID TUMOUR Hybrid tumours contain zones classic for more than one type of tumour histology. Oncocytic hybrid tumours contain areas with cells histologically consistent with oncocytoma, areas consistent with chromophobe RCC and mixed areas. SYNTENY In this context, this term is used to refer to gene loci in different organisms that are located on a chromosomal region of common evolutionary ancestry. BHD. Birt Hogg Dubé (BHD) syndrome, named after the three Canadian physicians who first described the dermatological features 108, is an autosomal-dominant genodermatosis that predisposes individuals to FIBROFOLLICULOMAS on the face and neck, SONTANEOUS NEUMOTHORAX and/or lung cysts, and an increased risk of renal neoplasia (TABLE 2). henotypic manifestations vary within and between families. A strong connection between the BHD skin lesions and renal manifestations was made when members of several families with inherited renal oncocytomas were identified with the classic BHD hallmark, fibrofolliculoma 109,110.In a large risk-assessment study of family members affected with BHD skin lesions, a 7-fold increased risk for development of renal tumours and a 50-fold increased risk for spontaneous pneumothorax were found 111.Although some have reported a co-association of BHD with colon polyps or colon cancer in other families 112,no increased risk for colon manifestations was identified in this study 111. Renal tumours found in individuals with BHD can be multifocal and bilateral, or unilateral and single, and display various histological features, including CHROMOHOBE RCC, oncocytoma, clear-cell RCC and even, rarely, papillary RCC (TABLE 2). However,the most frequent renal tumours are chromophobe RCCs and ONCOCYTIC HYBRID TUMOURS comprising elements of both oncocytomas and chromophobe RCCs 113,114. Microscopic oncocytosis found in the renal parenchyma indicated that these lesions might be precursors of hybrid oncocytic tumours, chromophobe renal carcinomas and perhaps clear-cell renal carcinomas in individuals with BHD syndrome. As individuals with large renal tumours have died of metastatic RCC, the renal manifestations of the syndrome can not be considered benign 113. Using fibrofolliculomas as an indicator of affected status, the BHD locus was identified on chromosome 17p11.2 by linkage 115,116, and mutations that co-segregated in BHD families were identified in a new gene 7. BHD encodes folliculin named for the fibrofolliculomas seen in BHD patients. Folliculin has no significant homology to any known human proteins although it is highly conserved across species. Almost all BHD mutations are insertions, deletions or nonsense mutations, predicted to truncate or prematurely terminate folliculin 7,112.Interestingly, nearly half of the mutations occur in a mononucleotide tract of cytosines Transcription of HIF target genes HIF-1 HIF-1 p300/cb HRE Figure 3 Fumarate-hydratase-inactivating mutations in hereditary leiomyomatosis renal-cell carcinoma lead to papillary type-2 renal-cell carcinoma. It is likely that impaired mitochondrial function due to fumarate hydratase (FH)-inactivating mutations (star) which block the conversion of fumarate to malate by the Krebs cycle lead to severe energy deficits (depletion of AT) and the formation of oxygen free radicals (O 2 ). This is sensed by the mitochondria as hypoxia, which leads to stabilization of HIF-1 subunits (see Fig. 1 for details) and transcriptional upregulation of hypoxia-inducible genes such as vascular endothelial growth factor, erythropoietin, platelet-derived growth factor- and transforming growth factor These proteins promote cell proliferation that could activate tumour growth. thought to be hypermutable because of slippage of the DNA polymerase during replication 117. Several animal models of BHD have been reported. The Nihon rat RCC model develops hereditary renal cancer, which is inherited in an autosomal-dominant manner with complete penetrance by 6 months of age. The gene locus was mapped by linkage analysis to chromosome 10p, which is SYNTENIC with human chromosome 17p11.2 (REFS ), and a disease-associated insertion mutation in the first coding exon of the rat Bhd orthologue was identified 120.Most renal tumours displayed LOH at the Bhd locus and the homozygous mutant condition resulted in embryonic lethality. Canine hereditary multifocal renal cystadenocarcinoma and nodular dermatofibrosis (RCND) is a naturally occurring inherited cancer syndrome in German-Shepherd dogs that is characterized by bilateral, multifocal renal tumours and numerous firm skin nodules made up of dense collagen fibres 121.The RCND locus maps to canine chromosome 5, which corresponds to human chromosome 17p11.2 (REF. 122).A missense mutation that changes a highly conserved histidine to arginine was found in the canine Bhd orthologue, which co-segregated with disease in the canine families with RCND and was not found in 264 control dogs 123.Homozygosity of the mutation is embryonically lethal in dogs with RCND, as in the Nihon rat. NATURE REVIEWS CANCER VOLUME 4 MAY

8 MICROSATELLITE INSTABILITY Describes diploid tumours in which genetic instability is due to a high mutation rate, primarily in short nucleotide repeats. Cancers with the microsatellite instability phenotype are associated with defects in DNA mismatchrepair genes. HAMARTOMAS Tumours comprising cells of more than one histological type. In contrast to VHL, BHD is infrequently mutated in sporadic renal tumours in humans, as several studies show a mutation rate of less than 10% 124,125.Although 17% 36% of sporadic renal tumours show LOH at the BHD locus and 11 33% show hypermethylation of the BHD promoter, depending on their histology 124,126,only one case with both somatic BHD mutation and LOH has been reported 125 indicating that BHD probably has only a minor role in the development of sporadic RCC. One study also reported LOH at the BHD locus in 81% of colorectal carcinomas and another detected a low frequency of BHD mutations in a set of colon carcinoma samples with MICROSATELLITE INSTABILITY 125,127,128.Further clarification of the role of BHD in colorectal carcinoma will require additional family studies. The function of BHD in normal cells and how protein-truncating mutations in folliculin lead to renal tumours, fibrofolliculomas and spontaneous pneumothorax remain to be established. Functional studies of the BHD protein have not been reported and therefore its role in the cell is unclear. Studies of BHD orthologues indicate a tumour-suppressor role for BHD, further supported by the high frequency of inactivating mutations found in the germline of individuals with BHD. Identification of second-hit mutations in BHD-associated renal tumours would confirm a tumour-suppressor function for folliculin. HT-JT. HT-JT is a rare autosomal dominantly inherited syndrome, which predisposes individuals to develop multiple parathyroid adenomas and multiple fibroosseous tumours of the jaw 129,130.Affected individuals from some families present with renal disease, including cystic kidney disease, HAMARTOMAS,mesoblastic nephromas, late-onset Wilms tumours and, in one patient, papillary renal-cell carcinoma 131,132.Recently, germline mutations were identified in a new gene HRT2 on chromosome 1q, which are predicted to cause deficient or impaired protein function 8.Associated LOH of the wildtype chromosome in the region of 1q21 q32, which is found in renal hamartomas 131, and frequent biallelic inactivation of HRT2 in sporadic parathyroid tumours 133,134 indicate that HRT2 acts as a tumour suppressor. The function of the HRT2 protein parafibromin is now under investigation. Hereditary syndromes with unknown genes Breakpoint genes. At least seven families that carry a constitutional chromosome-3 translocation have been described 135.These individuals have an increased risk for developing bilateral multifocal RCC, most often with conventional (clear-cell) histology, and genes at or near the breakpoints of the two chromosomes involved might be involved in RCC development. The fragile histidine triad (FHIT) gene spans the chromosome-3 breakpoint that was first identified in the first translocation family reported by Cohen 8,136. This family harbours a t(3;8)(p14;q24) balanced translocation. FHIT colocalizes with the most common fragile site in the human genome FRA3B and its expression is reduced or lost in various neoplasms 137. FHIT suppression of tumorigenicity in FHIT-negative cells indicates a tumoursuppressor role 138 ;however,how FHIT contributes to tumour development remains to be established. The other breakpoint-spanning gene in this translocation family is TRC8,which is located on chromosome 8 and is similar to the Drosophila segment-polarity gene patched (ptc) 139. TCH, the human homologue of ptc,acts as a tumoursuppressor gene and is involved in basal-cell carcinoma and medulloblastoma. Drosophila Trc8 protein was found to interact with the Drosophila Vhl protein, presenting a possible common pathway for TRC8 and VHL in humans 140.Disruption of three novel genes, DIRC1 (2q33) 141,DIRC2 (3q21) 142,and DIRC3 (2q35) 143,has been identified in two other families that have translocations with breakpoints at t(2;3)(q33;q21) and t(2;3)(q35;q21). DIRC3 was shown to form fusion transcripts with HS- BA1,a JmjC-Hsp27 domain gene, and might affect chromatin remodelling or stress-response signals leading to renal-cell carcinoma. LSAM (1q32), an IgLON family member, and NORE1 (3q13), which is homologous to a family of RAS-binding proteins, were identified as breakpoint-spanning genes in a Japanese family with t(1;3)(q32.1;q13.3), and hypermethylation of promoter regions of both genes was reported in renal tumours of patients 144.However, causative roles for each of these translocation breakpoint genes in the development of RCC remain to be determined. Given the role of VHL in the development of clearcell renal tumours, several renal tumours from affected members of these families with the chromosome-3 translocation were tested for 3p loss and VHL mutations. Nearly all cases showed loss of the derivative chromosome 3, and VHL mutations were confirmed in 12 of 22 renal tumours 135,145.These molecular and cytogenetic results indicate a three-step model for development of renal carcinoma in these families with chromosome-3 translocations 11,145 : inheritance of a germline chromosome-3 balanced translocation; nondisjunctional loss of the derivative chromosome that carries the short arm of chromosome 3; and somatic mutation or hypermethylation of the remaining tumour-suppressor gene on 3p, such as VHL. It is possible that other tumour-suppressor genes on chromosome 3p, such as RASSF1A, which is commonly hypermethylated in clear-cell RCC, are involved in the development of the renal carcinomas that are seen in these families with chromosome-3 translocations. apillary thyroid carcinoma with associated papillary renal neoplasia. apillary thyroid carcinoma is usually sporadic, but evidence for a genetic predisposition occurs in about 5% of cases. FTC is characterized by autosomal-dominant inheritance with age-dependent partial penetrance and an association with benign nodular thyroid disease. Linkage to several loci has been reported. An unusually large three-generation family with FTC was recently described in which two affected members had multifocal papillary renal neoplasms and one affected member developed renal oncocytoma 12. mutation analysis and analysis of genetic linkage to chromosome 1q21 was negative. This ruled out 388 MAY 2004 VOLUME 4

9 ODDS RATIO The odds ratio is a way of comparing whether the probability of a certain event is the same for two groups, and is calculated using a 2 2 table. An odds ratio of one implies that an event is equally likely in both groups. An odds ratio greater than one implies that an event is more likely in the first group. An odds ratio less than one implies that the event is less likely in the first group. HRC and indicated that a new renal-cancer-associated gene could be responsible for this familial syndrome. FCRC and future renal cancer gene discovery. Eleven small families, each with 2 5 members affected with familial clear-cell renal carcinoma (FCRC) without VHL mutations have also been reported 146,147. Linkage to chromosome 3p was excluded in all eleven families, nine of which were screened and found to be negative for mutations in and CUL2, indicating that additional renal carcinoma genes must exist. A large populationbased study was undertaken in Iceland to assess the role of heredity in the development of RCC in this isolated country 148.An extensive genealogical database ascertained the relatedness of all individuals diagnosed with RCC over a period of 45 years; first-degree relatives of RCC patients (siblings and parents) were found to have a 2 3-fold higher risk of developing RCC compared with the general population. Interestingly, a similar 2.5-fold ODDS RATIO for those with first-degree relatives affected by RCC was noted in a population from the western United States 149,strongly supporting a genetic component in common non-vhl RCC. An extensive recruitment effort is now underway in the United States for families with two or more individuals affected with non-vhl RCC for the purpose of mapping cancersusceptibility genes for familial RCC (W.M. Linehan and B. Zbar, personal communication). Diagnostic considerations There are no generally accepted screening guidelines for hereditary RCC syndromes; however, some recommendations can be made. A hereditary predisposition to renal cancer should be suspected whenever an individual who is diagnosed with renal cancer has a close relative also diagnosed with the disease, and/or when an individual presents with multifocal renal tumours or a history of previous renal tumour. Family history should be obtained and a pedigree created, paying specific attention to relatives with a known history of cancer. The relatively well-characterized syndromes VHL, HRC, HLRCC, BHD and HT-JT are all inherited in an autosomaldominant fashion. VHL, HLRCC, BHD, HT-JT and FTC have non-renal manifestations, whereas other syndromes, such as HRC, FCRC and chromosome-3 translocation, generally do not. Therefore, a thorough physical examination, focusing on skin, ophthalmological, neurological, parathyroid and thyroid abnormalities should be carried out. HLRCC and BHD are associated with dermatological lesions that can be observed by astute clinicians. Abdomino-pelvic computed tomography or magnetic resonance imaging with and without intravenous contrast medium are recommended for all individuals with suspected primary renal tumour, as ultrasound is insensitive for renal masses, particularly HRC 150.Multifocal and bilateral renal tumours are commonly found in RCC associated with hereditary syndromes (with the exception of HLRCC), and extra-renal manifestations of disease such as uterine tumours in HLRCC, pancreatic cysts or tumours, or adrenal pheochromocytomas in VHL might also be noted. If an RCC syndrome is not clearly evident after considering family history, physical examination and abdominal imaging, an effective way to tailor subsequent diagnostic studies is to obtain a pathological diagnosis of renal tumour type. Histological type varies between hereditary tumour syndromes; for example, in VHL and HRC a characteristic histology is noted in all cases (TABLE 2). So, histological differences between multiple renal tumours from the same individual virtually rule out VHL and HRC, but suggest BHD. Careful pathological re-review of all tumours resected, as well as tumours resected from first-degree relative(s) can help focus subsequent testing for specific syndromes. For example, if a genodermatosis (HLRCC or BHD) is suspected, skin biopsies and a high-resolution chest computed-tomography scan to look for lung cysts or evidence of previous pneumothoraces are recommended. If VHL is suspected, standard practice includes central nervous system imaging and biochemical testing for pheochromocytoma. The cloning of causative genes for VHL, HRC, HLRCC, BHD and HT-JT has made definitive genetic testing a reality for most syndromes that predispose to RCC. Germline genetic testing requires evaluation and referral by a certified genetic counsellor. At present, VHL screening and karyotyping are performed at numerous diagnostic laboratories in the United States, and tests for germline, FH and HRT2 mutations are now available (for further information, see the GeneTests web site in the online links box). The following recommendations can tentatively be made at this time. If the diagnostic criteria for VHL are met (for a review, see REF. 151), then germline VHL analysis is recommended. Genetic analysis of the VHL locus has demonstrated VHL inactivation in 100% of patients presenting with VHL by clinical criteria 152.Families with a documented predisposition to clear-cell RCC should be screened for germline VHL abnormalities and karyotyped to rule out chromosome-3 translocations. If papillary RCCs have been detected in a familial setting, then the sub-type papillary type 1 or type 2 must be determined histologically so that appropriate genetic testing can be considered: mutational screening of exons of for HRC and mutational analyses of FH for HLRCC (enzymatic-activity assays at present, but mutational analyses should be available soon). Genetic screening for BHD should be considered once this test becomes available. Any patient with hybrid oncocytic neoplasms, multifocal chromophobe RCC, and/or oncocytomas should ultimately be tested for mutations in BHD, as should families with discordant renal tumour histologies within or between family members. Genetic screening of at risk relatives, that is, firstdegree relatives of patients suspected to have hereditary renal neoplasia, should only occur once the proband has been found to have a heritable genetic anomaly causative of renal cancer. In cases where germline genetic testing is not available (for example, in BHD, FCRC and FTC), an updated search for such tests, followed by a history and physical examination of first-degree relatives are required if suspicion is high for a syndrome. These individuals should also be offered abdominal imaging at this time. NATURE REVIEWS CANCER VOLUME 4 MAY

10 VHL HIF-1 26S proteosome degradation HIF-1 Replace VHL gene, protein or function HRE HIF-1 HIF-1 Inhibit HIF-1 activity p300/cb Nucleus Transcription of HIF target genes Inhibit one or more HIF-induced gene products Figure 4 ossible targets for VHL-related therapeutics. Targeting the von Hippel Lindau (VHL) pathway for therapeutic intervention can theoretically occur at many sites. VHL protein function could be replaced, restoring binding to hypoxia-inducible factor-1 (HIF-1) and allowing its proteasomal degradation. The activity of HIF-1 could be a target for inhibition. Finally, molecules upregulated by HIF-1 also provide specific targets for potential downstream inhibition of the VHL pathway. Follow-up imaging scans should be obtained at regular intervals for carriers of germline predispositions to renal cancer. These studies are generally obtained every 6 12 months depending on the growth rate of a patient s previous or existing tumours. Implications for the treatment of RCC Individuals diagnosed with hereditary RCC should be considered for nephron-sparing treatment regimens, as these individuals are at risk of recurrent renal tumours throughout their lifetime. Such regimens include regular observation for most hereditary RCC tumours less than 3 cm in diameter 153,percutaneous ablation of renal tumours by radiofrequency 154 (heat) or cryotherapy 155 (cold), and/or nephron-sparing surgical approaches such as partial nephrectomy 156.Regular observation of patients with hereditary forms of RCC, followed by the removal of all lesions in the same kidney once a single lesion reaches greater than 3 cm, has been instituted by the National Cancer Institute in an attempt to minimize the number of operations an affected individual must have during his/her lifetime. Observation should include bi-yearly to yearly imaging, depending on the tumour growth rates. Very few deaths from metastatic RCC in families with VHL, HRC and BHD have been noted using this strategy, which has led to fewer surgeries in individuals affected by recurrent hereditary renal tumours 153. HLRCC is an exception, as RCC in these patients is often large and already metastatic at presentation. Surgical therapy is therefore recommended when HLRCC tumours are detected at any size before metastasis has occurred. Future directions Hereditary cancer syndromes often represent uncommon germline genetic abnormalities, which are similar to more common somatic abnormalities that cause sporadic neoplasia. This is well demonstrated for colon cancer, where hereditary cancer syndromes have led to the discovery of genes involved in sporadic colon cancer, and the sequential inactivation of different genes and genetic instability result in the malignant degeneration of precancerous lesions 157.For renal cancer, the paradigm that the germline abnormality in hereditary syndromes is commonly found in sporadic tumours is true for VHL in most, and for in some, cases of RCC. Further research is needed to determine whether the other syndrome-related genes discussed in this review contribute to sporadic RCC. This line of investigation would determine whether the progression of RCC depends on sequential alterations in distinct genes or whether RCC truly comprises distinct tumour types each with a classic mutation or set of genetic alterations, as might be predicted from most histological, molecular and cytogenetic analyses. So far, only a handful of renal tumours have been described where inactivation of more than one renal cancer tumour-suppressor gene has been found (BHD and VHL), indicating that tumours possibly progress from a more benign to a more malignant histology 113. A key goal in clinical oncology is the development of medical therapies specific to pathways that are altered in cancer. Understanding the biological pathways involving VHL,, FH, BHD and HRT2 will provide new therapeutic approaches for kidney cancer that were unimaginable before the identification of these target genes. Although the oncogenic pathways resulting from HRT2 and BHD mutation remain unknown, and those indicated by FH mutation remain speculative 106, much information is known about the pathways downstream of VHL and. Several groups have shown that VHL and are part of a common signalling pathway that is deregulated in RCC. Hypoxia activates transcription and amplifies HGF signalling to promote -dependent invasive growth 158.Expression of wild-type VHL in renal carcinoma cell lines inhibits HGF-induced invasion and branching morphogenesis by increasing levels of tissue inhibitors of metalloproteinases 159.Tumour invasion and metastasis requires dysregulation and degradation of the tissue matrix in cancer cells, and this complex process has been associated with HGF/ signalling. The specific targeting of such pathways in the hope of preventing RCC in highrisk individuals or medically treating existing RCC is the main goal of kidney oncology. Specific therapeutic options for VHL-related neoplasia involve restoring VHL protein function, inhibiting HIF activity, and/or targeting the downstream pathways that are activated by HIF (FIG. 4). Therapeutic gene replacement is a future possibility for VHL syndrome and for any syndrome that involves an inactivated tumour suppressor, possibly FH, BHD and HRT2.As the hypoxia pathway is activated in clear-cell RCC, several trials have already targeted angiogenesis in individuals with metastatic RCC. These include attempted therapy with thalidomide 160 and the VEGF-receptor inhibitor SU5416 (REF. 161), and a successful trial using an anti-vegf antibody 162.Current trials of imatinib 390 MAY 2004 VOLUME 4

11 (Glivec), an inhibitor of the BCR ABL fusion gene product that also inhibits HIF-upregulated plateletderived growth factor, and of EGF receptor inhibitors are also underway for clear-cell RCC. inhibitors are also being studied. SU11274 is a small-molecule inhibitor of that competitively binds its AT pocket and has had encouraging in vitro results; SU11274 adversely affects cellular growth in a dose-dependent fashion. Autophosphorylation of was reduced and the inhibitor blocked phosphorylation of AKT, glycogen synthase kinase-3 and the pro-apoptotic transcription factor FKHR 163.The drug geldanamycin has also shown in vitro effects against expression and its downstream effects 164.The development of agents that inactivate tyrosine kinase activity could provide future targeted therapies for oncogene-related renal tumour syndromes like HRC. Ultimately, the study of families with increased rates of cancer will continue to yield more insight into the factors that increase cancer risk. Genetic predispositions in the form of mutations and polymorphisms will increasingly be catalogued and DNA-level genetic profiling of high-risk families and individuals will become commonplace. Gene-expression profiling of renal tumours and of affected individuals should help to identify those individuals that benefit most from molecule-specific cancer therapies, as has been elegantly shown for haematopoietic malignancies and melanoma 165,166.Such work in renal neoplasia has already generated expression profiles for the common renal cancer histologies 167 and expression profiles with prognostic relevance for conventional (clear-cell) RCC 168,169.Current immunotherapeutic approaches to systemic treatment of advanced RCC are likely to benefit from the wealth of new molecular information obtained from such studies until more effective therapies are developed. Taken together, the advent of better diagnostic and discriminatory tools for both the hereditary and sporadic forms of renal cancer will no doubt improve the prognosis of diseases that, at present, are almost always lethal once metastatic. The increase in availability of genetic testing and counselling for high-risk families should prove both helpful and cost-effective, as genetically unaffected family members are reassured regarding their health status and removed from lifelong follow-up screening programmes. 1. Zbar, B., Klausner, R. & Linehan, W. M. Studying cancer families to identify kidney cancer genes. Annu. Rev. Med. 54, Chow, W. H., Devesa, S. S., Warren, J. L. & Fraumeni, J. F. Jr. Rising incidence of renal cell cancer in the United States. JAMA 281, (1999). 3. Kovacs, G. et al. The Heidelberg classification of renal cell tumours. J. athol. 183, (1997). 4. Latif, F. et al. Identification of the von Hippel Lindau disease tumor suppressor gene. Science 260, (1993). Describes the identification and cloning of the VHL tumour-suppressor gene and intragenic mutations in members of families with VHL. 5. Schmidt, L. et al. Germline and somatic mutations in the tyrosine kinase domain of the proto-oncogene in papillary renal carcinomas. Nature Genet. 16, (1997). First report of activating mutations in the protooncogene in inherited human cancer and identification of the causative gene for HRC. 6. Tomlinson, I.. et al. Germline mutations in FH predispose to dominantly inherited uterine fibroids, skin leiomyomata and papillary renal cell cancer. Nature Genet. 30, Identification of germline inactivating mutations in FH that predispose to HLRCC with papillary type-2 renal carcinoma. 7. Nickerson, M. L. et al. Mutations in a novel gene lead to kidney tumors, lung wall defects, and benign tumors of the hair follicle in patients with the Birt Hogg Dubé syndrome. Cancer Cell 2, Identification of germline protein-truncating mutations in a novel gene, BHD, in families with BHD syndrome leading to renal neoplasia of various histologies. 8. Carpten, J. D. et al. HRT2, encoding parafibromin, is mutated in hyperparathyroidism-jaw tumor syndrome. Nature Genet. 32, First report of germline inactivating mutations in a novel tumour-suppressor gene, HRT2, in families with HT-JT syndrome and associated renal neoplasia. 9. Cohen, A. J. et al. Hereditary renal-cell carcinoma associated with a chromosomal translocation. N. Engl. J. Med. 301, (1979). First report of a family with a balanced translocation involving chromosome 3 and a predisposition to renal-cell carcinoma. 10. Kovacs, G., Brusa,. & De Riese, W. Tissue-specific expression of a constitutional 3;6 translocation: development of multiple bilateral renal-cell carcinomas. Int. J. Cancer 43, (1989). 11. Bodmer, D. et al. An alternative route for multistep tumorigenesis in a novel case of hereditary renal cell cancer and a t(2;3)(q35;q21) chromosome translocation. Am. J. Hum. Genet. 62, (1998). 12. Malchoff, C. D. et al. apillary thyroid carcinoma associated with papillary renal neoplasia: genetic linkage analysis of a distinct heritable tumor syndrome. J. Clin. Endocrinol. Metab. 85, (2000). 13. Lendvay, T. S. & Marshall, F. F. The tuberous sclerosis complex and its highly variable manifestations. J. Urol. 169, Krymskaya, V.. Tumour suppressors hamartin and tuberin: intracellular signalling. Cell Signal. 15, Dome, J. S. & Coppes, M. J. Recent advances in Wilms tumor genetics. Curr. Opin. ediatr. 14, Maher, E. R. & Kaelin, W. G. Jr. von Hippel-Lindau disease. Medicine 76, (1997). 17. Chen, F. et al. Germline mutations in the von Hippel-Lindau disease tumor suppressor gene: correlations with phenotype. Hum. Mutat. 5, (1995). 18. Zbar, B. et al. Germline mutations in the Von Hippel-Lindau disease (VHL) gene in families from North America, Europe, and Japan. Hum. Mutat. 8, (1996). 19. Crossey,. A. et al. Molecular genetic investigations of the mechanism of tumourigenesis in von Hippel-Lindau disease: analysis of allele loss in VHL tumours. Hum. Genet. 93, (1994). 20. Herman, J. G. et al. Silencing of the VHL tumor-suppressor gene by DNA methylation in renal carcinoma. roc. Natl Acad. Sci. USA 91, (1994). 21. Vortmeyer, A. O. et al. Somatic point mutation of the wildtype allele detected in tumors of patients with VHL germline deletion. Oncogene 21, Lubensky, I. A. et al. Allelic deletions of the VHL gene detected in multiple microscopic clear cell renal lesions in von Hippel-Lindau disease patients. Am. J. athol. 149, (1996). 23. Mandriota, S. J. et al. HIF activation identifies early lesions in VHL kidneys: evidence for site-specific tumor suppressor function in the nephron. Cancer Cell 1, Foster, K. et al. Somatic mutations of the von Hippel- Lindau disease tumour suppressor gene in non-familial clear cell renal carcinoma. Hum. Mol. Genet. 3, (1994). 25. Gnarra, J. et al. Mutations of the VHL tumour suppressor gene in renal carcinoma. Nature Genet. 7, (1994). First demonstration of VHL mutation and LOH in most of sporadic clear-cell renal-cell carcinomas, which is the most common form of renal cancer. 26. Shuin, T. et al. Frequent somatic mutations and loss of heterozygosity of the von Hippel-Lindau tumor suppressor gene in primary human renal cell carcinomas. Cancer Res. 54, (1994). 27. Kaelin, W. G. Jr. Molecular basis of the VHL hereditary cancer syndrome. Nature Rev. Cancer 2, Kaelin, W. G. Jr. The von Hippel-Lindau gene, kidney cancer, and oxygen sensing. J. Am. Soc. Nephrol. 14, Maxwell,. HIF-1: an oxygen response system with special relevance to the kidney. J. Am. Soc. Nephrol. 14, Kim, W. & Kaelin, W. G. Jr. The von Hippel-Lindau tumor suppressor protein: new insights into oxygen sensing and cancer. Curr. Opin. Genet. Dev. 13, ugh, C. W. & Ratcliffe,. J. The von Hippel-Lindau tumor suppressor, hypoxia-inducible factor-1 (HIF-1) degradation, and cancer pathogenesis. Semin. Cancer Biol. 13, Maxwell,. H. et al. The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis. Nature 399, (1999). Landmark publication that demonstrated an interaction between VHL and HIF proteins, and stability of HIF- subunits in VHL-deficient cell lines. 33. Semenza, G. L. Regulation of mammalian O 2 homeostasis by hypoxia-inducible factor 1. Annu. Rev. Cell Dev. Biol. 15, (1999). 34. Bruick, R. K. & McKnight, S. L. A conserved family of prolyl- 4-hydroxylases that modify HIF. Science 294, Stebbins, C. E., Kaelin, W. G. Jr., & avletich, N.. Structure of the VHL-Elongin C-Elongin B complex: implications for VHL tumor suppressor function. Science 284, (1999). Crystal structure of VHL protein in complex with elongins B and C that identified the frequently mutated - and -domains. 36. ause, A. et al. The von Hippel-Lindau tumor-suppressor gene product forms a stable complex with human CUL-2, a member of the Cdc53 family of proteins. roc. Natl Acad. Sci. USA 94, (1997). 37. Hon, W. C. et al. Structural basis for the recognition of hydroxyproline in HIF-1 by pvhl. Nature 417, Min, J. H. et al. Structure of an HIF-1-pVHL complex: hydroxyproline recognition in signaling. Science 296, Ivan, M. et al. HIF targeted for VHL-mediated destruction by proline hydroxylation: implications for O 2 sensing. Science 292, This paper and reference 40 demonstrated that the VHL HIF interaction is regulated by oxygendependent prolyl hydroxylation. 40. Jaakkola. et al. Targeting of HIF- to the von Hippel- Lindau ubiquitylation complex by O 2 -regulated prolyl hydroxylation. Science 292, NATURE REVIEWS CANCER VOLUME 4 MAY

12 41. Cockman, M. E. et al. Hypoxia inducible factor- binding and ubiquitylation by the von Hippel-Lindau tumor suppressor protein. J. Biol. Chem. 275, (2000). 42. Ohh, M. et al. Ubiquitination of hypoxia-inducible factor requires direct binding to the -domain of the von Hippel- Lindau protein. Nature Cell Biol. 2, (2000). 43. Lando, D., eet, D. J., Whelan, D. A., Gorman, J. J. & Whitelaw, M. L. Asparagine hydroxylation of the HIF transactivation domain: a hypoxic switch. Science 295, Mahon,. C., Hirota, K. & Semenza, G. FIH-1: a novel protein that interacts with HIF-1 and VHL to mediate repression of HIF-1 transcriptional activity. Genes Dev. 15, Iliopoulos, O., Kibel, A., Gray, S. & Kaelin, W. G. Jr. Tumor suppression by the human von Hippel-Lindau gene product. Nature Med. 1, (1995). This was the first report to identify the VHL gene product in cells and the first to show that restoration of wild-type VHL function in VHL / renal carcinoma cells suppressed the formation of tumours in nude mice. 46. Kondo, K., Klco, J., Nakamura, E., Lechpammer, M. & Kaelin, W. G. Jr. Inhibition of HIF is necessary for tumor suppression by the von Hippel-Lindau protein. Cancer Cell 1, The creation of a stable HIF mutant prevents tumour suppression by VHL in a nude mouse model, implicating the VHL HIF interaction as critical to the growth of renal cancer cells. 47. Kondo, K., Kim, W. Y., Lechpammer, M. & Kaelin, W. G., Jr. Inhibition of HIF2 is sufficient to suppress pvhl-defective tumor growth. LOS Biol. 1, Maranchie, J. K. et al. The contribution of VHL substrate binding and HIF1- to the phenotype of VHL loss in renal cell carcinoma. Cancer Cell 1, VHL substrate binding at the HIF recognition domain of VHL protein was shown to be essential to the tumour-suppressor function of VHL, but artificially increasing HIF levels alone was not sufficient for tumorigenesis. 49. Gnarra, J. R. et al. ost-transcriptional regulation of vascular endothelial growth factor mrna by the product of the VHL tumor suppressor gene. roc. Natl Acad. Sci. USA 93, (1996). 50. Levy, A.., Levy, N. S., & Goldberg, M. A. Hypoxia-inducible protein binding to vascular endothelial growth factor mrna and its modulation by the von Hippel-Lindau protein. J. Biol. Chem. 271, (1996). 51. Iliopoulos, O., Levy, A.., Jiang, C., Kaelin, W. G. Jr & Goldberg, M. A. Negative regulation of hypoxia-inducible genes by the von Hippel-Lindau protein. roc. Natl Acad. Sci. USA 93, (1996). 52. Siemeister, G. et al. Reversion of deregulated expression of vascular endothelial growth factor in human renal carcinoma cells by von Hippel-Lindau tumor suppressor protein. Cancer Res. 56, (1996). 53. Knebelmann, B., Ananth, S., Cohen, H. T. & Sukhatme, V.. Transforming growth factor is a target for the von Hippel- Lindau tumor suppressor. Cancer Res. 58, (1998). 54. de aulsen, N. et al. Role of transforming growth factor- in von Hippel Lindau (VHL) / clear cell renal carcinoma cell proliferation: a possible mechanism coupling VHL tumor suppressor inactivation and tumorigenesis. roc. Natl Acad. Sci. USA 98, Ohh, M. et al. The von Hippel-Lindau tumor suppressor protein is required for proper assembly of an extracellular fibronectin matrix. Mol. Cell 1, (1998). 56. Chen, F. et al. Suppression of growth of renal carcinoma cells by the von Hippel-Lindau tumor suppressor gene. Cancer Res. 55, (1995). 57. ause, A., Lee, S., Lonergan, K. M., & Klausner, R. D. The von Hippel-Lindau tumor suppressor gene is required for cell cycle exit upon serum withdrawal. roc. Natl Acad. Sci. USA 95, (1998). 58. Davidowitz, E. J., Schoenfeld, A. R. & Burk, R. D. VHL induces renal cell differentiation and growth arrest through integration of cell-cell and cell-extracellular matrix signaling. Mol. Cell. Biol. 21, Bindra, R. S., Vasselli, J. R., Stearman, R., Linehan, W. M. & Klausner, R. D. VHL-mediated hypoxia regulation of cyclin D1 in renal carcinoma cells. Cancer Res. 62, Baba, M. et al. Loss of von Hippel-Lindau protein causes cell density dependent deregulation of CyclinD1 expression through hypoxia-inducible factor. Oncogene 22, Staller,. et al. Chemokine receptor CXCR4 downregulated by von Hippel-Lindau tumour suppressor pvhl. Nature 425, Zbar, B. et al. Germline mutations in the Von Hippel-Lindau disease (VHL) gene in families from North America, Europe, and Japan. Hum. Mutat., 8, (1996). 63. Friedrich, C. A. Genotype-phenotype correlation in von Hippel-Lindau syndrome. Hum. Mol. Genet. 10, Clifford, S. C. et al. Contrasting effects on HIF-1 regulation by disease-causing pvhl mutations correlate with patterns of tumourigenesis in von Hippel-Lindau disease. Hum. Mol. Genet. 10, Hergovich, A., Lisztwan, J., Barry, R., Ballschmieter,. & Krek, W. Regulation of microtubule stability by the von Hippel-Lindau tumour suppressor protein pvhl. Nature Cell Biol. 5, Hoffman, M. A. et al. von Hippel-Lindau protein mutants linked to type 2C VHL disease preserve the ability to downregulate HIF. Hum. Mol. Gen. 10, Feldman, D. E., Spiess, C., Howard, D. E. & Frydman, J. Tumorigenic mutations in VHL disrupt folding in vivo by interfering with chaperonin binding. Mol. Cell 12, Maranchie, J. K. et al. Solid renal tumor severity in von Hippel Lindau disease is related to germline deletion length and location. Hum. Mutat. 23, (2004). 69. Gnarra, J. R. et al. Defective placental vasculogenesis causes embryonic lethality in VHL-deficient mice. roc. Natl Acad. Sci. USA 94, (1997). 70. Haase, V. H., Glickman, J. N., Socolovsky, M. & Jaenisch, R. Vascular tumors in livers with targeted inactivation of the von Hippel-Lindau tumor suppressor. roc. Natl Acad. Sci. USA 98, Ma, W. et al. Hepatic vascular tumors, angiectasis in multiple organs, and impaired spermatogenesis in mice with conditional inactivation of the VHL gene. Cancer Res. 63, Zbar, B. et al. Hereditary papillary renal cell carcinoma: clinical studies in 10 families. J. Urol. 153, (1995). 73. Schmidt, L. et al. Two North American families with hereditary papillary renal carcinoma and identical novel mutations in the proto-oncogene. Cancer Res. 58, (1998). 74. Schmidt, L. et al. Novel mutations of the protooncogene in papillary renal carcinomas. Oncogene 18, (1999). 75. Olivero, M. et al. Novel mutation in the AT-binding site of the oncogene tyrosine kinase in a HRCC family. Int. J. Cancer 82, (1999). 76. Bottarro, D.. et al. Identification of the hepatocyte growth factor receptor as the c-met proto-oncogene product. Science 251, (1991). A molecule upregulated after treatment of target cells in vitro with HGF was found to be identical to the c- proto-oncogene product, now known as the HGF transmembrane tyrosine kinase receptor. 77. Bardelli, A., ugliese, L., & Comoglio,. M. Invasive growth signalling by the Met/HGF receptor. The hereditary renal carcinoma connection. Biochim. Biophys. Acta. 1333, M41 M51 (1997). 78. Comoglio,. M. athway specificity for Met signaling. Nature Cell Biol. 3, E161 E Birchmeier, C., Birchmeier, W., Gherardi, E. & Vande Woude, G. F. Met, metastasis, motility and more. Nature Rev. Mol. Cell Biol. 4, Trusolino, L. & Comoglio,. M. Scatter-factor and semaphorin receptors: cell signalling for invasive growth. Nature Rev. Cancer 2, Jeffers, M. et al. Activating mutations for the met tyrosine kinase receptor in human cancer. roc. Natl Acad. Sci. USA 94, (1997). 82. Hofstra, R. M. et al. A mutation in the RET proto-oncogene associated with multiple endocrine neoplasia type 2B and sporadic medullary thyroid carcinoma. Nature 367, (1994). 83. Nagata, H. et al. Identification of a point mutation in the catalytic domain of the protooncogene c-kit in peripheral blood mononuclear cells of patients who have mastocytosis with an associated hematologic disorder. roc. Natl Acad. Sci. USA 92, (1995). 84. Shu, H. K., elley, R. J. & Kung, H. J. Tissue-specific transformation by epidermal growth factor receptor: a single point mutation within the AT-binding pocket of the erbb product increases its intrinsic kinase activity and activates its sarcomagenic potential. roc. Natl Acad. Sci. USA 87, (1990). 85. Miller, M. et al. Structural basis of oncogenic activation caused by point mutations in the kinase domain of the proto-oncogene: modeling studies. roteins 44, Michieli,. et al. Mutant Met-mediated transformation is ligand-dependent and can be inhibited by HGF antagonists. Oncogene 18, (1999). 87. Chiara, F., Michieli,., ugliese, L. & Comoglio,. M. Mutations in the met oncogene unveil a dual switch mechanism controlling tyrosine kinase activity. J. Biol. Chem. 278, Zhuang, Z. et al. Trisomy 7-harbouring non-random duplication of the mutant allele in hereditary papillary renal carcinomas. Nature Genet. 20, (1998). 89. Fischer, J. et al. Duplication and overexpression of the mutant allele of the proto-oncogene in multiple hereditary papillary renal cell tumours. Oncogene 17, (1998). 90. Schmidt, C. et al. Scatter factor/hepatocyte growth factor is essential for liver development. Nature 373, (1995). 91. Uehara, Y. et al. lacental defect and embryonic lethality in mice lacking hepatocyte growth factor/scatter factor. Nature 373, (1995). 92. Bladt, F., Riethmacher, D., Isenmann, S., Aguzzi, A. & Birchmeier, C. Essential role for the c-met receptor in the migration of myogenic precursor cells into the limb bud. Nature 376, (1995). 93. Jeffers, M. et al. The mutationally activated Met receptor mediates motility and metastasis. roc. Natl Acad. Sci. USA 95, (1998). 94. Liang, T. J., Reid, A. E., Xavier, R., Cardiff, R. D. & Wang, T. C. Transgenic expression of tpr-met oncogene leads to development of mammary hyperplasia and tumors. J. Clin. Invest. 97, (1996). 95. Launonen, V. et al. Inherited susceptibility to uterine leiomyomas and renal cell cancer. roc. Natl Acad. Sci. USA 98, Kiuru, M. et al. Familial cutaneous leiomyomatosis is a twohit condition associated with renal cell cancer of characteristic histopathology. Am. J. athol. 159, Delahunt, B. et al. Morphologic typing of papillary renal cell carcinoma: comparison of growth kinetics and patient survival in 66 cases. Hum. athol. 32, Jiang, F. et al. Chromosomal imbalances in papillary renal cell carcinoma: genetic differences between histological subtypes. Am. J. athol. 153, (1998). 99. Toro, J. et al. Mutations in the fumarate hydratase gene cause hereditary leiomyomatosis and renal cell cancer in families in North America. Am. J. Hum. Genet. 73, Alam, N. A. et al. Genetic and functional analyses of FH mutations in multiple cutaneous and uterine leiomyomatosis, hereditary leiomyomatosis and renal cancer, and fumarate hydratase deficiency. Hum. Mol. Genet. 12, Alam, N. A. et al. Localization of a gene (MCUL1) for multiple cutaneous leiomyomata and uterine fibroids to chromosome 1q42. 3-q43. Am. J. Hum. Genet. 68, Kiuru, M. et al. Few FH mutations in sporadic counterparts of tumor types observed in hereditary leiomyomatosis and renal cell cancer families. Cancer Res. 62, Bevan, S. et al. Germline mutations in fumarate hydratase (FH) do not predispose to prostate cancer. rostate Cancer rostatic Dis. 6, Barker, K. T. et al. Low frequency of somatic mutations in the FH/multiple cutaneous leiomyomatosis gene in sporadic leiomyosarcomas and uterine leiomyomas. Br. J. Cancer 87, Coughlin, E. M. et al. Molecular analysis and prenatal diagnosis of human fumarase deficiency. Mol. Genet. Metab. 63, (1998) Eng, C., Kiuru, M., Fernandez, M. J. & Aaltonen, L. A. A role for mitochondrial enzymes in inherited neoplasia and beyond. Nature Rev. Cancer 3, Vanharanta, S. et al. Early-onset renal cell carcinoma as a novel extraparaganglial component of SDHB-associated heritable paraganglioma. Am. J. Hum. Genet. 74, (2004) Birt, A. R., Hogg, G. R. & Dubé, W. J. Hereditary multiple fibrofolliculomas with trichodiscomas and acrochordons. Arch. Dermatol. 113, (1977) Weirich, G. et al. Familial renal oncocytoma: clinicopathological study of 5 families. J. Urol. 160, (1998) Toro, J. et al. Birt Hogg Dubé syndrome: a novel marker of kidney neoplasia. Arch. Dermatol. 135, (1999) Zbar, B. et al. Risk of renal and colonic neoplasms and spontaneous pneumothorax in the Birt Hogg Dubé syndrome. Cancer Epidemiol. Biomarkers rev. 11, An attempt to define BHD as a cancer-predisposition syndrome from its humble origins as a simple genodermatosis Khoo, S. K. et al. Clinical and genetic studies of Birt Hogg Dubé syndrome. J. Med. Genet. 39, MAY 2004 VOLUME 4

13 113. avlovich C.. et al. Renal tumors in the Birt Hogg Dubé syndrome. Am. J. Surg. athol. 26, Tickoo, S. K. et al. Renal oncocytosis: a morphologic study of fourteen cases. Am. J. Surg. athol. 23, (1999) Schmidt, L. S. et al. Birt Hogg Dubé syndrome, a genodermatosis associated with spontaneous pneumothorax and kidney neoplasia, maps to chromosome 17p Am. J. Hum. Genet. 69, Khoo, S. K. et al. Birt Hogg Dubé syndrome: mapping of a novel hereditary neoplasia gene to chromosome 17p12- q Oncogene 20, Streisinger, G. et al. Frameshift mutations and the genetic code. Cold Spring Harbor Symp. Quant. Biol. 31, (1966) Okimoto, K. et al. A novel Nihon rat model of a Mendelian dominantly inherited renal cell carcinoma. Jpn J. Cancer Res. 91, (2000). A spontaneous and now established rat model of renal cancer similar to human BHD that may be a valuable research tool. Also see references 119 and Hino, O., Okimoto, K., Kouchi, M. & Sakurai, J. A novel renal carcinoma predisposing gene of the Nihon rat maps on chromosome 10. Jpn J. Cancer Res. 92, Okimoto, K. et al. A germ-line insertion in the Birt Hogg Dubé (BHD) gene gives rise to the Nihon rat model of inherited renal cancer. roc. Natl Acad. Sci. USA 101, (2004) Lium, B. & Moe, L. Hereditary multifocal renal cystadenocarcinomas and nodular dermatofibrosis in the German shepherd dog: macroscopic and histopathologic changes. Vet. athol. 22, (1985) Jonasdottir, T. J. et al. Genetic mapping of a naturally occurring hereditary renal cancer syndrome in dogs. roc. Natl Acad. Sci. USA 97, (2000) Lingaas, F. et al. A mutation in the canine BHD gene is associated with hereditary multifocal renal cystadenocarcinoma and nodular dermatofibrosis in the German Shepherd dog. Hum. Mol. Genet. 12, The first natural large animal model for renal-cell carcinoma Khoo, S. K. et al. Inactivation of BHD in sporadic renal tumors. Cancer Res. 63, da Silva, N. F. et al. Analysis of the Birt Hogg Dubé (BHD) tumour suppressor gene in sporadic renal cell carcinoma and colorectal cancer. J. Med. Genet. 40, avlovich, C.. et al. Genetic analysis of renal cell carcinomas in Birt Hogg Dubé (BHD) syndrome. roc. Am. Assoc. Cancer Res. 43, Shin, J. H. et al. Mutations of the Birt Hogg Dubé (BHD) gene in sporadic colorectal carcinomas and colorectal carcinoma cell lines with microsatellite instability. J. Med. Genet. 40, Kahnoski, K. et al. Alterations of the Birt Hogg Dubé gene (BHD) in sporadic colorectal tumours. J. Med. Genet. 40, Jackson, C. E. et al. Hereditary hyperparathyroidism and multiple ossifying jaw fibromas: a clinically and genetically distinct syndrome. Surgery 108, (1990) Szabo, J. et al. Hereditary hyperparathyroidism-jaw tumor syndrome: the endocrine tumor gene HRT2 maps to chromosome 1q21-q31. Am. J. Hum. Genet. 56, (1995) Teh, B. T. et al. Autosomal dominant primary hyperparathyroidism and jaw tumor syndrome associated with renal hamartomas and cystic kidney disease: linkage to 1q21-q32 and loss of the wild type allele in renal hamartomas. J. Clin. Endocrinol. Metab. 81, (1996) Haven, C. J. et al. A genotypic and histopathological study of a large Dutch kindred with hyperparathyroidism-jaw tumor syndrome. J. Clin. Endocrinol. Metab. 85, (2000) Shattuck, T. M. et al. Somatic and germ-line mutations of the HRT2 gene in sporadic parathyroid carcinoma. N. Engl. J. Med. 349, Howell, V. M. et al. HRT2 mutations are associated with malignancy in sporadic parathyroid tumours. J. Med. Genet. 40, Bodmer, D. et al. Understanding familial and non-familial renal cell cancer. Human Mol. Genet. 11, Ohta, M. et al. The FHIT gene, spanning the chromosome 3p14. 2 fragile site and renal carcinoma-associated t(3;8) breakpoint, is abnormal in digestive tract cancers. Cell 84, (1996) Ong, S. T. et al. recise localization of the FHIT gene to the common fragile site at 3p14. 2 (FRA3B) and characterization of homozygous deletions within FRA3B that affect FHIT transcription in tumor cell lines. Genes Chromosom. Cancer 20, (1997) Siprashvili, Z. et al. Replacement of Fhit in cancer cells suppresses tumorigenicity. roc. Natl Acad. Sci. USA 94, (1997) Gemmill, R. M. et al. The hereditary renal cell carcinoma 3;8 translocation fuses FHIT to a patched-related gene, TRC8. roc. Natl Acad. Sci. USA 95, (1998) Gemmill, R. M. et al. The TRC8 hereditary kidney cancer gene suppresses growth and functions with VHL in a common pathway. Oncogene 21, Druck, T. et al. The DIRC1 gene at chromosome 2q33 spans a familial RCC-associated t(2;3)(q33;q21) chromosome translocation. J. Hum. Genet. 46, Bodmer, D. et al. Disruption of a novel MFS transporter gene, DIRC2, by a familial renal cell carcinoma-associated t(2;3)(q35;q21). Hum. Mol. Genet. 11, Bodmer, D., Schepens, M., Eleveld, M. J., Schoenmakers, E. F. & Geurts van Kessel, A. Disruption of a novel gene, DIRC3, and expression of DIRC3-HSBA1 fusion transcripts in a case of familial renal cell cancer and t(2;3)(q35;q21). Genes Chromosom. Cancer 38, Chen, J. et al. The t(1;3) breakpoint-spanning genes LSAM and NORE1 are involved in clear cell renal cell carcinomas. Cancer Cell 4, Schmidt, L. et al. Mechanism of tumorigenesis of renal carcinomas associated with the constitutional chromosome 3;8 translocation. Cancer J. Sci. Am. 1, (1995) Woodward, E. R., Clifford, S. C., Astuti, D., Affara, N. A. & Maher, E. R. Familial clear cell renal cell carcinoma (FCRC): clinical features and mutation analysis of the VHL,, and CUL2 candidate genes. J. Med. Genet. 37, (2000) Teh, B. T. et al. Familial non-vhl non-papillary clear cell renal cancer. Lancet 349, (1997) Gudbjartsson, T. et al. A population-based familial aggregation analysis indicates genetic contribution in a majority of renal cell carcinomas. Int. J. Cancer 100, Shows that the future of gene discovery for RCC could rely on the discovery of predispositions that are more subtle than those found so far, which have been autosomal dominant syndromes following classic Mendelian inheritance Gago-Dominguez, M., Yuan, J. M., Castelao, J. E., Ross, R. K. & Yu, M. C. Family history and risk of renal cell carcinoma. Cancer Epidemiol. Biomarkers rev. 10, Choyke,. L. Imaging of hereditary renal cancer. Radiol. Clin. North Am. 41, Lonser, R. R. et al. von Hippel-Lindau disease. Lancet 361, Stolle, C. et al. Improved detection of germline mutations in the von Hippel-Lindau disease tumor suppressor gene. Hum. Mutat. 12, (1998) Herring, J. C. et al. arenchymal sparing surgery in patients with hereditary renal cell carcinoma: 10-year experience. J. Urol. 165, Along with reference 156, a demonstration that nephron-sparing approaches are appropriate for patients with syndromes placing them at continued risk of renal neoplasia avlovich, C.. et al. ercutaneous radiofrequency ablation of small renal tumors: initial results. J. Urol. 167, Shingleton, W. B. & Sewell,. E. Jr. ercutaneous renal cryoablation of renal tumors in patients with von Hippel- Lindau disease. J. Urol. 167, Steinbach, F. et al. Treatment of renal cell carcinoma in von Hippel-Lindau disease: a multicenter study. J. Urol. 153, (1995) Grady, W. M. Genetic and epigenetic alterations in colon cancer. Annu. Rev. Genomics Hum. Genet. 3, ennacchietti, S. et al. Hypoxia promotes invasive growth by transcriptional activation of the met protooncogene. Cancer Cell 3, Koochekpour, S. et al. The von Hippel-Lindau tumor suppressor gene inhibits hepatocyte growth factor/scatter factor-induced invasion and branching morphogenesis in renal carcinoma cells. Mol. Cell Biol. 19, (1999) Amato, R. J. Thalidomide therapy for renal cell carcinoma. Crit. Rev. Oncol. Hematol. 46, S59 S Lara,. N. Jr et al. SU5416 plus interferon in advanced renal cell carcinoma: a phase II California Cancer Consortium Study with biological and imaging correlates of angiogenesis inhibition. Clin. Cancer Res. 9, Yang, J. C. et al. A randomized trial of bevacizumab, an antivascular endothelial growth factor antibody, for metastatic renal cancer. N. Engl. J. Med. 349, Sattler, M. et al. A novel small molecule met inhibitor induces apoptosis in cells transformed by the oncogenic TR- tyrosine kinase. Cancer Res. 63, Webb, C.. et al. The geldanamycins are potent inhibitors of the hepatocyte growth factor/scatter factor-met-urokinase plasminogen activator-plasmin proteolytic network. Cancer Res. 60, (2000) Alizadeh, A. A. et al. Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling. Nature 403, (2000) Bittner, M. et al. Molecular classification of cutaneous malignant melanoma by gene expression profiling. Nature 406, (2000) Higgins, J.. et al. Gene expression patterns in renal cell carcinoma assessed by complementary DNA microarray. Am. J. athol. 162, Takahashi, M. et al. Gene expression profiling of clear cell renal cell carcinoma: gene identification and prognostic classification. roc. Natl Acad. Sci. USA 98, Vasselli, J. R. et al. redicting survival in patients with metastatic kidney cancer by gene-expression profiling in the primary tumor. roc. Natl Acad. Sci. USA 100, Acknowledgements This publication has been funded in part with Federal funds from the National Cancer Institute, National Institutes of Health. The content of this publication does not reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products or organizations imply endorsement by the United States Government. Competing interests statement The authors declare that they have no competing financial interests. Online links DATABASES The following terms in this article are linked online to: Cancer.gov: BHD CB CUL2 CXCR4 cyclin D1 DIRC1 DIRC2 DIRC3 elongin B elongin C EO FH FHIT FIH-1 HGF HIF-1 HIF-1 HIF-2 HIF-3 HRT2 LSAM NORE1 p300 TCH RASSF1A TGF- TRC8 VEGF VHL LocusLink: colon cancer renal-cell carcinoma OMIM: Birt Hogg Dubé syndrome familial papillary thyroid carcinoma hereditary leiomyomatosis and renal-cell cancer hereditary papillary renal carcinoma hyperparathyroidism-jaw tumour syndrome multiple cutaneous and uterine leiomyomatosis tuberous sclerosis Wilms tumour FURTHER INFORMATION GeneTests web site: National Society Of Genetic Counselors: Access to this interactive links box is free online. NATURE REVIEWS CANCER VOLUME 4 MAY