In 2003, malignancy was the second leading cause of mortality after heart disease in the USA, representing ≈ 23% of all deaths that year . Currently, kidney cancer affects an estimated 39 000 individuals in the USA and is responsible for nearly 13 000 deaths annually . Surgical resection is recommended for most patients with localized tumours, while for those with advanced disease, immunotherapy regimens offer 10–20% response rates. The study of the hereditary types of kidney cancer syndromes has helped to elucidate the genetic basis of kidney tumorigenesis, and provided the basis for developing molecular-targeted agents in the treatment of this disease.
RCC represents distinct pathological entities that are distinguishable by histological subtype: clear cell RCC (75%), type 1 papillary RCC (5%), type 2 papillary RCC (10%), chromophobe RCC and oncocytoma (10%) (Fig. 1) . Specific genetic alterations are linked to the histological subtypes. In this review we examine the inherited forms of kidney cancer and how the genetic understanding of these hereditary kidney cancers facilitated the development of targeted therapeutic strategies in the treatment of kidney cancer.
VON HIPPEL-LINDAU (VHL)
VHL was initially reported in 1904 by Eugene von Hippel and later described by Arvid Lindau in 1926 [3,4]. Affected individuals are at risk of developing tumours in the retina, cerebellum, spinal cord, inner ear, pancreas, adrenal glands, kidneys and epididymis. VHL is inherited in an autosomal dominant pattern and affects 1 in 35 000 individuals in the USA. To identify the VHL disease gene, families with several members showing the clinical manifestations of VHL were evaluated at the National Cancer Institute (NCI). Studies of families with hereditary renal cancer showing germline translocations involving the short arm of chromosome 3 prompted an analysis of restriction fragment length polymorphisms in tumour tissue from patients with sporadic RCC . That study showed a non-random loss of heterozygosity (LOH) on the short arm of chromosome 3 in tumour tissue from patients with sporadic, nonhereditary RCC. This led to the hypothesis that a null mutation on the corresponding locus of the homologous chromosome 3 might be important in the development of renal cancer . In 1993, Latif et al. reported a linkage analysis on 221 VHL kindred to identify the VHL gene on a small region of the distal portion of chromosome 3p. The identification of VHL somatic mutations in clear cell RCC, with the lack of other chromosomal deletions in localized renal tumours, strongly implicated the VHL gene in th7e origin or early development of renal tumours . This conclusion was further supported by Lubensky et al., who found LOH in the VHL gene in microscopic tumours and in atypical cysts in kidneys from VHL patients. In cases where no mutation was identified, hypermethylation in the 5′ region of the VHL gene was found to be an alternative mechanism for inactivation of gene expression . The link between the VHL mutation in the germline and the clinical manifestations of the syndrome was subsequently confirmed by the identification of a spectrum of germline mutations in 99% of VHL families [10,11].
Evidence that the VHL gene acts as a tumour suppressor gene came from studies in which tumours from patients with sporadic RCC were found to have a mutation in one allele and a deletion of the second allele, consistent with Knudsen’s ‘two hit’ hypothesis [8,12,13]. Subsequent studies helped define the function of the VHL gene and the genetic pathway to tumorigenesis after VHL mutation. Duan et al. found that the VHL gene product forms a complex with elongin C and elongin B. This complex was then shown to bind to cul-2, which gave the first insight into VHL as an e3 ubiquitin ligase . Further work showed that the VHL complex targets the α subunits of hypoxia-inducible factors (HIF1α and HIF2α) for ubiquitin-mediated degradation, and that inactivation of the VHL gene by mutation or methylation resulted in the accumulation of HIF, which in turn resulted in the activation of vascular endothelial growth factor (VEGF), epidermal growth factor, TGF and glucose transporter (Glut-1) pathways [16,17,17–19]. Kondo et al. and Maranchie et al. reported studies suggesting that HIF2 is a critical pathway in clear cell RCC tumorigenesis (Fig. 2) .
Of patients with VHL, ≈ 60% develop solid and cystic renal lesions during their lifetime, resulting in significant morbidity and potential mortality. Before the widespread use of CT, metastatic RCC was the leading cause of death among VHL patients . Pathological examination of the renal tumours showed that they are well-circumscribed lesions with clear cell histology. The kidneys of affected patients often contain numerous solid and cystic lesions. After examining the normal renal parenchyma, Poston et al. and Walther et al. estimated that the average VHL kidney contains ≈ 1100 cysts (benign or atypical), and ≈ 600 clear cell neoplasms. The primary goal in managing the renal manifestations of VHL is to prevent metastatic disease. At the NCI, close surveillance with serial cross-sectional imaging and nephron-sparing surgery are used to control the risk of metastasis and maintain native renal function whenever possible [26,27]. VHL patients with solid renal lesions are often followed until the lesions reach 3 cm, when nephron-sparing surgery is recommended. In a study of 181 VHL patients managed with this strategy, no patient with tumours of ≤ 3 cm developed metastatic disease or end-stage renal disease requiring dialysis .
Patients with VHL are also prone to developing phaeochromocytomas, which can be multiple, bilateral, extra-adrenal, and, occasionally, malignant. Historically, hereditary adrenal phaeochromocytomas were treated with complete adrenalectomy; however, as patients developed additional lesions over time, surgical resection left patients with no functioning adrenal tissue. At the NCI, VHL patients with adrenal phaeochromocytomas are managed predominantly with partial adrenalectomy (open or laparoscopic). In a recent study of 33 patients (43 tumours) who had laparoscopic partial adrenalectomy, at mean follow-up of 36 months, only two had evidence of recurrence (both subsequently resected laparoscopically) and one required long-term adrenal replacement therapy [29,30].
HEREDITARY PAPILLARY RENAL CARCINOMA (HPRC)
In 1994, Zbar et al. described three families with many affected members developing type 1 papillary RCC. The disorder was not linked to polymorphic markers on chromosome 3p, and there was no LOH at the VHL gene locus. The authors concluded that a different gene might be involved in papillary renal tumorigenesis. Similar to the investigation of VHL, families were recruited and genetic linkage analysis used which identified a responsible locus on chromosome 7q31, corresponding to the Met proto-oncogene . Subsequent experiments identified missense mutations in the tyrosine kinase domain of Met in the germline of affected patients, suggesting that the mutation led to constitutive activation of the Met protein and the development of papillary RCC . The Met gene encodes a cell membrane receptor specific for hepatocyte growth factor (HGF). In vitro studies showed that HGF stimulation leads to mitogenesis, cellular migration and morphogenesis . Analysis of additional families with HPRC allowed the identification of novel mutations in the gene, as well as the role of trisomy 7 in papillary tumorigenesis. Schmidt et al. studied sporadic papillary RCC tumours and identified c-met mutations in 13% of tumours. Evaluation of both HPRC tumours and sporadic papillary tumours revealed that tumours with c-met mutations have a distinctive papillary phenotype (papillary type 1 RCC) that was genetically and histologically different from renal tumours in other hereditary renal cancer syndromes, and from most sporadic renal tumours with papillary architecture . These results suggested that non-inherited papillary renal cancers might develop by different mechanisms from those in HPRC (Fig. 3).
Individuals from families with HPRC are at risk of developing bilateral, multifocal type 1 papillary RCC [36,37]. Given the autosomal dominant inheritance pattern, and highly penetrant phenotype, patients are at risk of developing up to 3000 tumours in each kidney. Tumours tend to develop later in life, at median age of 50–70 years, although a recent report identified three families with early-onset HPRC. Affected members of these families developed tumours at 19–35 years old and several individuals developed metastasis . This report highlighted the potential for metastasis in a syndrome with tumours that are predominantly well differentiated. At the NCI, HPRC patients with renal tumours are managed with a strategy similar to VHL. Tumours of <3 cm are followed expectantly while those >3 cm are treated with nephron-sparing surgery. Choyke et al. studied the imaging characteristics of family members of HPRC and noted that ultrasonography detected only 45% of the lesions visualized on CT. Furthermore, HPRC-associated tumours had lower enhancement characteristics (by Hounsfield units) than a comparable group of clear cell RCC tumours. Thus, contrast-enhanced CT or MRI with gadolinium are appropriate imaging methods for screening individuals from HPRC-affected families.
BHD was first describe by Birt et al. in 1977, as a genodermatosis characterized by an autosomal dominant inheritance pattern in which affected individuals develop cutaneous papules on the face, neck and upper trunk. Roth et al. then reported a patient with BHD who had developed bilateral, multifocal chromophobe renal tumours. These reports prompted the recruitment of families with BHD to characterize the phenotypic manifestations of the syndrome, and the risk of renal tumours. In a study of 150 individuals from 49 families, Toro et al. identified three extended families in whom the manifestations of BHD and renal tumours segregated together, but in whom no member was found to have germline mutations in the VHL or Met genes. Also notable were four individuals from two of the families who had pulmonary cysts and/or spontaneous pneumothorax. These findings pointed to a novel familial renal cancer syndrome gene; and subsequently, genetic linkage analyses led to the mapping of the BHD gene to chromosome 17p11.2 . Mutational analysis from BHD families revealed that the alteration in the gene predicted truncation of the BHD protein (folliculin) . Vocke et al. used DNA sequencing analysis of BHD tumours and discovered frameshift mutations in the second copy of the gene that predicted truncation of the BHD protein. These results strongly suggested a role of the BHD protein (folliculin) as a tumour-suppressor gene that predisposed to renal tumours once both copies were inactivated. Further laboratory investigations are ongoing to elucidate the pathways that lead to tumorigenesis after alteration of the gene (Fig. 4)
Of affected patients with BHD, 15–30% develop kidney tumours. In a review of 30 patients with 130 BHD associated renal tumours, Pavlovich et al. reported a remarkable diversity of histological subtypes: 34% chromophobe RCC, 50% hybrid oncocytic RCC, 9% clear cell RCC, 5% oncocytoma, and 2% papillary RCC. In addition, evaluation of the normal parenchyma revealed multiple areas of renal oncocytosis that are putative precursor lesions, suggesting that the entire kidney is at risk of tumours. In general, renal tumours associated with BHD tend to have a lower propensity for metastasis than clear cell RCC, but have been known to metastasize . To preserve renal function, patients with BHD are also managed conservatively at the NCI, with nephron-sparing surgery reserved for tumours of >3 cm. After cataloguing clinical manifestations of 98 patients affected with BHD, Zbar et al. reported an age-adjusted odds ratio for spontaneous pneumothorax of 50.3 in affected individuals. Patients with BHD should be screened for pulmonary cysts and counselled about the signs and symptoms of spontaneous pneumothorax (Fig. 5).
HEREDITARY LEIOMYOMATOSIS AND RENAL CELL CANCER (HLRCC)
Although Reed et al. in 1973 initially described a syndrome characterized by cutaneous leiomyomas and uterine leiomyomas, it was Launonen et al. in 2001 who first described a new familial kidney cancer syndrome, named HLRCC. In an initial description of two European families with the syndrome, genetic linkage analysis led to the identification of the HLRCC gene, fumarate hydratase (FH) on chromosome 1q . Further analysis of renal tumours and a uterine leiomyoma showed LOH at the HLRCC gene locus . Toro et al. subsequently described genetic mutations in North American families with HLRCC. The HLRCC gene, FH, an enzyme in the tricarboxylic acid cycle, is integral in the oxidation of pyruvate for the production of cellular energy by catalysing the conversion of fumarate to malate. Family members with germline mutation in the FH have significant decreases in FH activity, and renal tumours from these patients have extremely low or absent FH activity . The biallelic loss shown in both uterine and renal tumours in these studies suggests a tumour-suppressor function of the gene; however, the specific tumorigenic pathways affected by the FH mutation are still unknown. Recently, Isaacs et al. reported on a link between FH inactivity and the up-regulation of HIF-dependent pathways. Under normoxic conditions, HIF1α and HIF2α are targeted for degradation by the VHL protein complex. VHL recognition of HIF requires the enzymatic hydroxylation of two proline residues on HIF catalysed by HIF prolyl hydroxylase. Isaacs et al. showed that excess intracellular fumarate interferes with HIF prolyl hydroxylase function, resulting in the accumulation of HIF and potentially, up-regulation of its downstream target genes, VEGF and Glut-1. Investigations are ongoing to search for other possible tumorigenic pathways involved in HLRCC (Fig. 6).
Affected family members with HLRCC develop cutaneous leiomyomas and large uterine leiomyomas, which often necessitate hysterectomy before 30 years old, or predispose to spontaneous abortions. By contrast with HPRC, HLRCC patients develop an aggressive type of RCC with papillary features. To date, >50 families with HLRCC have been evaluated at the NCI, which has facilitated the characterization of the clinical manifestations of the syndrome. Although there is significant variability in the expression of the cutaneous leiomyomas, >90% of female FH mutation carriers developed uterine leiomyomas .
Renal tumours occur less frequently in these families, but tend to be aggressive, and in many instances, lethal. In the initial two reports of 33 HLRCC families, 17 family members were identified with renal tumours, and of these 17 patients, 13 died from metastatic disease within 5 years of diagnosis [49,52]. Unlike the other familial kidney cancer syndromes, HLRCC-associated renal tumours can be solitary and unilateral. At the NCI, suspected HLRCC patients are evaluated with contrast-enhanced CT or gadolinium-enhanced MRI. Unlike patients with other familial renal cancer syndromes, HLRCC patients with renal tumours are not followed expectantly; furthermore, given the potential aggressive nature of these renal tumours, a more aggressive surgical approach is often recommended.
TARGETED THERAPIES FOR KIDNEY CANCER
Although the discovery of renal cancer genes came from studies of patients with familial renal cancer syndromes, investigations of sporadic tumours with similar histological characteristics have shown similar genetic alterations in many cases. Of sporadic clear cell RCC tumours, ≈ 75% show loss of VHL activity and 13% of sporadic papillary type 1 tumours have activating mutations in the Met proto-oncogene. The gradual elucidation of the mechanisms by which these genetic alterations lead to tumorigenesis has provided the basis for the development of pathway-targeted therapeutics. In clear cell RCC, constitutive activation of the HIF pathway results in increased transcription of platelet-derived growth factor (PDGF) and VEGF, leading to cell proliferation and angiogenesis, which are thought to be associated with the development and growth of tumours.
Sunitinib malate and sorafenib are both oral multi-targeted tyrosine kinase inhibitors that selectively block segments of HIF-dependent angiogenesis pathways. Both drugs were recently approved for treating patients with advanced kidney cancer. In a randomized phase III trial, Motzer et al. reported a 34% partial response rate and 8.3 month median progression-free survival in 106 patients with metastatic kidney cancer treated with 50 mg of sunitinib. In addition, in a randomized phase II trial, 73 of 202 patients with metastatic kidney cancer treated with sorafenib had tumour shrinkage of ≥ 25% and a longer median progression-free survival than patients treated with placebo . Similar studies are underway investigating potential inhibitors of the c-Met pathway, both in the laboratory and as early clinical trials in patients with both hereditary and sporadic papillary RCC. It is hoped that identifying additional tumorigenic pathways in patients with BHD and HLRCC will elucidate targets for novel therapeutic agents to treat hereditary and sporadic tumours with related histology.
This research was supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.