Review

Correspondence *Urologic Oncology Branch, National Cancer Institute, 10 Center Drive MSC 1107, Building 10 CRC, Room 1-5940, Bethesda, MD 20892-1107, USA

Email linehanm@mail.nih.gov

Introduction

It is now well established that kidney cancer does not represent a single disease, but rather a collection of different types of cancers. Significant insight into the molecular pathogenesis of these cancers has been gained through the study of families with multiple members affected with kidney malignancies. Evaluation of these families has led to the identification of genes involved in the development of a number of different types of kidney cancer. Previously identified familial forms of kidney cancer include von Hippel?Lindau disease (VHL), hereditary papillary renal carcinoma, and Birt?Hogg?Dub?syndrome. All three disorders are associated with a genetic predisposition to the formation of kidney cancers that results from an inherited, germline mutation. Linkage studies of affected families have led to the identification of the causative gene in each of these clinical entities.

A hereditary form of kidney cancer referred to as hereditary leiomyomatosis and renal cell cancer (HLRCC) has been identified, in which affected family members have cutaneous leiomyomas, uterine fibroids, and/or kidney cancers.¹ The renal malignancies that develop in HLRCC families are often metastatic at presentation and are a significant cause of mortality in these families. Analysis of families with this disorder has identified the responsible gene locus as FH.² This gene encodes fumarate hydratase (FH), an enzyme that is part of the mitochondrial Krebs or tricarboxylic acid (TCA) cycle. The mechanism by which alterations in FH lead to HLRCC remains to be determined, but it apparently involves increased cellular dependence on glycolysis. This  summarizes the currently known clinical, genetic, and molecular aspects of HLRCC, to assist the clinician in the detection and management of this potentially life-threatening disorder.

Clinical Manifestations of HLRCC

Similar to patients with other forms of hereditary kidney cancer such as VHL, hereditary papillary renal carcinoma, and Birt?Hogg?Dub?syndrome, patients with HLRCC are at risk of developing tumors in multiple organs, namely the skin, uterus, and kidney. The identification of cutaneous leiomyomas in the context of familial kidney cancer is highly indicative of HLRCC. Cutaneous leiomyomas are highly penetrant in HLRCC families. In a study that reported on the second cohort of North American HLRCC families evaluated by the National Cancer Institute, cutaneous leiomyomas were found in 76% of families.³ Patients with cutaneous manifestations tend to have multiple lesions. These lesions most often develop on the trunk and extremities (Figure 1). Furthermore, the lesions are often symptomatic: most patients (90%) complain of sensitivity to light touch, paresthesias, or pain.^{3, 4} Patients are young at the onset of cutaneous disease, with a mean age of onset of 25 years in one series.⁴ While the cutaneous lesions can be quite apparent, there are cases where patients might have a single cutaneous lesion or lesions that are asymptomatic.⁴ As a result, consultation with a dermatologist might be beneficial when considering a diagnosis of HLRCC. Furthermore, pathologic confirmation of suspected cutaneous leiomyoma is crucial to the diagnosis of HLRCC.

Figure 1 Clinical and pathologic manifestations of HLRCC.

(A) Cutaneous leiomyomas.²⁴ (B) MRI scan that shows uterine fibroids (arrows). (C) Large, left-sided renal mass from a young male with germline FH mutations.²⁴ (D) HLRCC-associated renal carcinoma is characterized by orangiophilic nucleoli.³⁷ Abbreviations: FH, fumarate hydratase gene; HLRCC, hereditary leiomyomatosis and renal cell cancer.

Similar to cutaneous manifestations, uterine leiomyomas (fibroids) are also highly penetrant in HLRCC families (Figure 1). Furthermore, the presence of cutaneous leiomyomas is highly predictive of the presence of fibroids in families with a known germline mutation of the FH gene. Toro et al. found that 98% of women with cutaneous leiomyomas had uterine fibroids.⁴ Symptoms that are characteristic of fibroids include menorrhagia or dysmenorrhea. While fibroids are also common in the general population, fibroid disease in women with HLRCC often has an earlier age of onset. In one study, almost 70% of women known to harbor FH mutations were diagnosed with fibroids at age 30 years or younger.³ In addition, female patients with HLRCC and fibroids often require intervention at a young age. Wei et al. found that 50% of women in their cohort who required surgery for fibroid disease had their surgery (either myomectomy or hysterectomy) at age 30 years or younger.³ In addition, a few cases of uterine leiomyosarcoma have been described in patients with known germline mutations of FH.^{1, 5} Nevertheless, the predominant uterine finding associated with HLRCC is leiomyoma, which can be a major source of morbidity in HLRCC-affected women.

While kidney tumors seem to be less penetrant in HLRCC families than uterine and skin leiomyomas, renal tumors are a serious clinical manifestation of HLRCC. While the association of cutaneous leiomyomas and uterine fibroids was established more than 30 years ago,⁶ the link with kidney cancer was only made in 2001.¹ Renal tumors have been identified in approximately one-third of the HLRCC families evaluated at the National Cancer Institute.³ While this proportion is higher than previously reported,⁷ differences in family recruitment and imaging techniques could account for the discrepancy.³ Most patients tend to have solitary renal lesions (Figure 1); however, bilateral, multifocal lesions have been identified. Most patients with HLRCC have kidney tumors with a distinct histology. When Merino et al. examined multiple kidney tumors from patients with HLRCC, they identified tumor nuclei that contained inclusion-like nucleoli that were orangiophilic, with a perinucleolar halo (Figure 1).⁸ These tumors can be aggressive and spread early. In the first cohort of North American families with HLRCC to be described, 13 individuals were affected with RCC. Of these 13, 9 had died from metastatic disease within 5 years of diagnosis.⁴ These findings underscore the importance of regular surveillance of patients with known germline mutations of the FH gene.

Genetics of HLRCC

As mentioned previously, the genetic alteration in HLRCC has been mapped to the FH gene located on the long (q) arm of chromosome 1.² The FH gene codes for FH (also known as fumarase). The FH gene contains 10 exons; its protein product exists as a homotetramer.³ This enzyme catalyzes the hydration of fumarate to form malate, a reaction that is part of the TCA cycle (Figure 2). HLRCC is transmitted in an autosomal-dominant fashion. The FH gene is thought to act as a tumor-suppressor gene, in that HLRCC carcinogenesis follows Knudson's 'two-hit' model (in which individuals inherit a predisposing mutation, but cancer does not develop until a second mutation occurs in the same gene).⁹

Figure 2 The tricarboxylic acid (TCA) cycle and hereditary cancer syndromes.

Metabolites of the TCA cycle are represented in the dark boxes, and enzymes that catalyze the steps of the cycle are shown in the light boxes. Mutations of SDH genes result in hereditary paraganglioma and mutations of FH result in HLRCC. Abbreviations: CoA, coenzyme A; FH, fumarate hydratase gene; HLRCC, hereditary leiomyomatosis and renal cell cancer; SDH, succinate dehydrogenase complex genes; TCA, tricarboxylic acid.

In the case of HLRCC, patients inherit a mutant copy of FH that is present in the germline. Tumor formation is associated with an alteration in the inherited wild-type allele, and (leiomyoma or kidney cancer) is thought to result from lack of wild-type FH. Loss of the wild-type allele (i.e. loss of heterozygosity) in HLRCC-associated tumors was examined by Tomlinson et al. Over 80% of the leiomyomas evaluated were found to have loss of heterozygosity in chromosome 1q.² Among the seven kidney tumors evaluated by this group, alterations of the wild-type allele included FH gene deletion (five tumors), partial deletion (one tumor), and missense (one tumor) mutations.² A clinical entity referred to as FH deficiency results from inheritance of two mutated copies of the FH gene. This is an autosomal-recessive disorder marked by severe encephalopathy.¹⁰

Genetic testing detects FH mutations in over 90% of HLRCC families.⁴ Germline alterations that have been detected include missense, nonsense, insertion, deletion, and splice-site mutations.^{2, 3} In their series of HLRCC families, Tomlinson et al. reported a high rate of mutations in genetic loci that corresponded to the amino portion of the FH protein;² however, mutation analysis of North American families revealed an even distribution of mutations throughout the FH gene.⁴ Furthermore, analysis of germline mutations in HLRCC patients with kidney cancer revealed that mutations were distributed throughout the FH gene.³ In addition, there was no apparent correlation between mutation location or type and the development of kidney cancer, as is seen in other forms of hereditary kidney cancer such as VHL.^{3, 11} No phenotype?genotype correlation could be detected with respect to either fibroids or cutaneous leiomyomas.⁷

Role of FH mutations in Cancer

The identification of germline gene alterations that result in hereditary forms of kidney cancer has shed light on the genetic basis of sporadic RCC. Alterations of the VHL gene have been found in a large percentage of sporadic, clear-cell renal neoplasms.^{12, 13} While it is clear that germline mutations are associated with the development of the kidney tumors that are part of the HLRCC complex, the role of FH alterations in sporadic kidney cancer remains to be determined. At this time, there is no conclusive evidence that somatic mutations of FH have a significant role in sporadic kidney cancers.^{5, 14}

Mechanism of tumor formation

The reason why FH alterations are associated with tumor formation in HLRCC families is not entirely clear at this time. It seems intuitive that a cell that lacks functional FH (and hence has a defective TCA cycle) would be at a metabolic disadvantage, particularly with regard to the efficiency of nutrient catabolism. HLRCC is not, however, the only hereditary cancer syndrome associated with a defective enzyme of the Krebs cycle. Germline mutations in the succinate dehydrogenase complex have been identified that predispose to the development of hereditary paragangliomas. Succinate dehydrogenase catalyzes the conversion of succinate to fumarate?the step in the TCA cycle that immediately precedes the reaction catalyzed by FH (Figure 2). Mutations in subunits B, C, and D of the succinate dehydrogenase complex have all been linked to hereditary paraganglioma.^{15, 16, 17}

Interestingly, three cases of RCC have been described in individuals with germline mutations of SDHB. In all three cases (two of the three patients were members of the same family), RCC was diagnosed when these individuals were in their 20s.¹⁸ The overlap between HLRCC and hereditary paraganglioma is intriguing, and indicates that similar mechanisms might drive tumor formation in these two distinct, inherited, neoplastic syndromes. Potential mechanisms of tumorigenesis identified in one syndrome might, therefore, also apply to the other. The principal mechanisms postulated to link FH mutations and tumorigenesis include diminished apoptosis and pseudohypoxia.¹⁹

Most of the evidence, so far, supports pseudohypoxia as the mode of tumorigenesis in HLRCC. Pseudohypoxia is best understood in the context of the VHL pathway. Alterations in the VHL pathway have been implicated in the development of both sporadic and hereditary clear-cell neoplasms (Figure 3). Intact VHL protein is known to form a complex with other proteins, including Cullin-2, elongin B, and elongin C, which together are referred to as the VHL complex.²⁰ The VHL complex possesses ubiquitin ligase activity that targets various proteins for proteasome-mediated degradation.^{21, 22, 23} Two of the proteins targeted by the VHL complex are hypoxia-inducible factors (HIFs) 1 and 2, which are key regulators of the cellular response to hypoxia.^{21, 22, 23} HIFs upregulate the transcription of several genes involved in angiogenesis, metabolism, and cell growth and/or proliferation, such as vascular endothelial growth factor (VEGF), glucose transporter 1 (GLUT1), platelet-derived growth factor (PDGF), transforming growth factor (TGF), and erythropoietin (EPO).²⁴ These downstream targets of HIFs are genes that might be important in carcinogenesis.²³

Figure 3 Under normoxic conditions HIF is hydroxylated by HPH.

Hydroxylated HIF is then recognized by the VHL complex and targeted for ubiquitin-mediated degradation. VHL mutations result in a pseudohypoxic state because HIF fails to be degraded and hence accumulates. The result is the upregulation of candidate carcinogenic genes. In the context of HLRCC, HIF accumulation can occur owing to inhibition of HPH by fumarate. When HPH is inhibited, HIF remains unhydroxylated and, therefore, avoids degradation. Abbreviations: EPO, erythropoietin gene; GLUT1, glucose transporter 1 gene; HIF, hypoxia-inducible factor; HLRCC, hereditary leiomyomatosis and renal cell cancer; HPH, HIF prolyl hydroxylase; O₂, oxygen; OH, hydroxyl group bound to HIF; PDGF, platelet-derived growth factor; TGF, gene for transforming growth factor ; VEGF, vascular endothelial growth factor gene; VHL, von Hippel?Lindau protein.

 

Under normoxic conditions, HIFs are hydroxylated by enzymes called HIF prolyl hydroxylases that add hydroxyl groups to proline residues in HIFs (Figure 3).^{25, 26, 27, 28, 29} In their hydroxylated form, HIF proteins are recognized by the VHL complex and are subsequently targeted for degradation by the proteasome. Under hypoxic conditions, HIFs fail to be hydroxylated by HIF prolyl hydroxylases, because oxygen is a required substrate.³⁰ In their unhydroxylated form, HIFs are not recognized by the VHL complex and hence avoid degradation; they are consequently available to upregulate the transcription of VEGF, GLUT1, PDGF, TGF and EPO. Pseudohypoxia, therefore, results from an aberration in the VHL pathway. In the context of clear-cell carcinoma (both sporadic and associated with VHL disease), HIFs avoid degradation because of the lack of a functional VHL complex, owing to mutation of the VHL gene (Figure 3). As a result, HIF levels and those of downstream targets of HIF are elevated.^{31, 32} The upregulation of VEGF in this scenario might explain why clear-cell cancers are vascular tumors.²⁴

Similarly, there are data that suggest tumorigenesis in HLRCC is associated with pseudohypoxia. There is increased HIF expression in tumors from HLRCC patients. Pollard et al. demonstrated (by immunochemistry and immunoblotting) that there was strong HIF-1 expression in kidney tumors from HLRCC patients.³³ Isaacs et al. demonstrated increased expression of both HIF-1 and HIF-2 in renal tumors from patients with HLRCC compared to normal, matched renal tissue from the same patient.³⁰ In addition, there is also evidence of increased expression of downstream targets of HIF. Pollard et al. found increased microvessel density in leiomyomas from HLRCC patients, when compared to matched normal myometrium from HLRCC patients.³⁴ Furthermore, the observed increase in microvessel density correlated with an increase in expression of VEGF (i.e. increased numbers of VEGF transcripts).³⁴

While there is evidence that pseudohypoxia drives HLRCC-associated tumorigenesis, the mechanisms by which this occurs have not been determined. Isaacs et al. have, however, shown that fumarate is an inhibitor of HIF prolyl hydroxylase.³⁰ In the setting of diminished FH activity, fumarate accumulation could occur, which would result in increased levels of unhydroxylated HIFs (Figure 3). Unhydroxylated HIFs avoid recognition by the VHL complex (and hence avoid ubiquitin-mediated degradation). The result is a pseudohypoxic state characterized by upregulation of genes that are potentially integral to carcinogenesis (Figure 3). Interestingly, succinate (the metabolite that is dehydrogenated to form fumarate in the TCA cycle) has also been shown to inhibit HIF prolyl hydroxylases, which could explain the mechanism of tumor formation in patients with germline mutations of SDH.³⁵

While the mechanisms of tumor formation in HLRCC have focused on the role of FH in the Krebs cycle, a cytosolic form of FH exists.³⁶ At this time, little is known about the function of cytosolic FH, or about a potential link to oncogenesis.

Clinical Management of Patients with HLRCC

The clinical management of patients with HLRCC requires an integrated, multidisciplinary approach. Diagnostic evaluation of individuals who are suspected of having HLRCC includes a thorough pedigree analysis. As mentioned earlier, genetic testing is available; however, it is not 100% reliable, and a clinical diagnosis can be based on an individual's and/or their family history. Particular attention is paid to the presence of leiomyomatous manifestations, as these are more highly penetrant in HLRCC families than kidney tumors. Nevertheless, a family history of renal cancer raises clinical suspicion. Dermatologic evaluation is important, as pathologic evaluation of suspected cutaneous leiomyomas aids in the diagnosis of HLRCC. Further dermatologic consultation could also be necessary as cutaneous leiomyomas can also be asymptomatic. Gynecologic evaluation and management is also integral to the evaluation of female patients, as HLRCC-affected women often have symptoms indicative of uterine fibroids.

Patients with HLRCC but without evidence of renal tumors are screened periodically (e.g. yearly) with imaging modalities such as CT or MRI. For those patients with indeterminate renal lesions, close follow-up is recommended with more frequent than annual imaging. Early intervention is advocated for patients with evidence of renal masses, as renal cancers in patients with HLRCC have a high malignant potential. Studies are in progress to determine the role of nephron-sparing surgery in patients affected with HLRCC-associated kidney cancer. No form of systemic therapy has been shown to be effective for patients with metastatic disease.

Conclusion

HLRCC represents a distinct clinical entity that confers a genetic predisposition to the development of cutaneous and uterine leiomyomas, as well as lethal kidney cancers. The causative genetic alterations have recently been mapped to the FH gene, which codes for an enzyme that is part of the Krebs cycle. While the molecular mechanism of pathogenesis is not entirely clear, pseudohypoxia is thought to be involved. The relatively recent characterization of this familial kidney cancer represents an increasing trend in urologic oncology to identify the biologic basis of cancer.

The accumulation of detailed information on this disorder at the clinical and molecular levels will provide an improved understanding of the pathogenesis of kidney cancers, both inherited and sporadic. With continued investigation into HLRCC as well as other forms of kidney cancer, the clinician should be better able to classify RCC patients according to the biologic basis of their tumors, in order to determine the optimal therapeutic regimen.

Acknowledgements

This research was supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.

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Competing interests

The authors declared no competing interests.