Seminars in Ophthalmology, 2013; 28(5–6): 377–386 ! Informa Healthcare USA, Inc. ISSN: 0882-0538 print / 1744-5205 online DOI: 10.3109/08820538.2013.825281

Von Hippel-Lindau Disease: A Genetic and Clinical Review1 Nour Maya N. Haddad1, Jerry D. Cavallerano1,2, and Paolo S. Silva1,2 1

Beetham Eye Institute, Joslin Diabetes Center, Boston, Massachusetts, USA and 2Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, USA

ABSTRACT Background: Von Hippel–Lindau Disease (VHL) is an autosomal dominant inherited systemic cancer syndrome that gives rise to cystic and highly vascularized tumors in many organs, including the eye. Recent studies have contributed to the understanding of VHL pathophysiology, genetics, and the role of the VHL protein. This article reviews recent studies on VHL clinical findings, genetics and tumorigenesis. Methods: Literature review of articles on VHL genetics with correlation to clinical findings. Results: Genotype-phenotype correlation studies show that patients with a complete deletion mutation of the VHL gene, relative to participants with a missense or protein-truncating mutation, had better visual acuity and decreased tumorigenesis incidence of retinal hemangioblastomas. It has also been documented that higher levels of vascular endothelial growth factor (VEGF), hypoxia induced factor (HIF), and ubiquitin are found in ocular hemangioblastomas. The stromal foamy vacuolated cells seem to be the true tumor cells of the disease acting on the surrounding endothelial cells in ocular hemangioblastomas. Tumor cells and ocular lesions have shown increased levels of Erythropoietin (Epo), Epo receptor (EpoR), and CD133. Also, CXCR4, a CXC chemokine receptor, is expressed in retinal VHL hemangioblastomas. Recent studies suggest that the VHL mutation alone may not be sufficient to develop VHL-associated neoplasms. Studies suggest that targeting various proteins along with anti-angiogenesis molecules may be a better therapeutic approach than targeting VEGF alone. Conclusion: Understanding of the mechanisms and genetics underlying VHL and its associated retinal hemangioblastomas has increased substantially in recent years. This knowledge suggests that future advances may include better identification of individuals at higher risk of vision loss and the development of novel individualized therapies. Keywords: Angiogenesis, genotype-phenotype correlation, hypoxia induced factor, optic disc hemangioblastoma, retinal hemangioblastoma, VEGF

INTRODUCTION

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ophthalmologist, first described retinal lesions (‘‘angiomatosis retinae’’) in 1904.3 Arvid Lindau, a Swedish pathologist, recognized the association between retinal and cerebellar hemangioblastomas in 19274 and also described the presence of visceral lesions. Patients with VHL are at increased risk of developing central nervous system and retinal hemangioblastomas, clear cell renal carcinoma, pheochromocytomas, neuroendocrine tumors and cysts of the pancreas, endolymphatic sac tumors, papillary

Von Hippel–Lindau (VHL) disease (OMIM 193300) occurs in approximately one per 36,000 live births per year. VHL syndrome is caused by a mutation in the VHL tumor suppressor gene. The disease is transmitted in an autosomal dominant mode and is highly penetrant. By the age of 65 years, more than 90% of individuals with VHL will display some diseaserelated symptoms.1 The average age of onset is in the third decade of life.2 Eugen von Hippel, a German

Received 28 April 2013; accepted 11 July 2013; published online 19 September 2013 1

The authors are grateful to Lloyd Paul Aiello, MD, Jan Lammer, MD, and Robert W. Cavicchi, CRA, FOPS, for assisting with the preparation of this review. Correspondence: Nour Maya Haddad, Beetham Eye Institute, Joslin Diabetes Center, 1 Joslin Place, Boston MA 02215, USA. E-mail: [email protected]

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378 N. M. N. Haddad et al. cystadenomas of the epididymis and broad ligament.5,6 Patients are classified as VHL type 1 when they have no pheochromocytomas and as VHL type 2 when pheochromocytomas are present. Type 2 patients are categorized into three subgroups: type 2A with low risk of renal cell carcinomas (RCC), type 2B with high risk of RCC, and type 2C with isolated pheochromocytoma.7–9 Retinal hemangioblastomas, present in up to 85% of individuals with VHL, are the most common lesion of VHL disease.10 VHL-associated retinal hemangioblastomas are usually found at a mean age of 25 years but the lesions can also occur in infancy.11 In this article, we review recent studies on VHL clinical findings, genetics and tumorigenesis, including HIFdependent and HIF-independent pathways.

MATERIAL AND METHODS A literature review was performed in the Medline databases in March 2013 to identify recent studies on VHL genetics, tumorigenesis, and clinical correlation. The individual or combined searched terms included Von Hippel-Lindau, genetics, hypoxia induced factor, physiopathology, genotype-phenotype correlation, angiogenesis, VEGF, VHL gene, and retinal or optic nerve head hemangioblastoma.

VHL Gene Location Von Hippel-Lindau disease is caused by germ-line mutations in the VHL tumor suppressor gene. The gene is denoted as von Hippel-Lindau tumor suppressor, E3 ubiquitin protein ligase. The gene is located on the short arm of chromosome 3 (3p25-26) (Figure 1), spans 10 kb, is composed of three exons, and encodes for two different protein isoforms (pVHL19 and pVHL30). The complete VHL protein consists of 214 amino acids and has two structural domains: the a-domain and the -domain.6

Role of the VHL Protein pVHL30 consists of 213 amino acids while pVHL19 contains residues 54–213 of the pVHL30. pVHL19 displays a high homology among human, dog, mouse, and rat tissue, and maintains all the functional domains of the pVHL30. The protein model of pVHL19, published in 1999 by Stebbins et al.,12 is composed of two functional subdomains, a smaller, helical a-domain, which is composed of three helices (H1, H2, and H3), and a larger -domain (residues 63– 154 and residues 193–204), which creates a sevenstranded sandwich and a helix (H4). Two important sites have been identified where most of the diseasecausing VHL gene mutations are located: the first represents the elongin C-binding site (amino acid residues 157–170) and the second is the hypoxiainducible factor 1 a (HIF1 a) binding site (amino acid residues 91–113 in the -domain).13 Therefore, pVHL is the substrate recognition unit of an E3 ubiquitin ligase complex that comprises Cul2, Elongin B and C, and Rbx1.13 Substrates of this complex are the a subunits of the heterodimeric transcription factor hypoxia-inducible factor (HIF), leading to its ubiquitylation and proteasome-mediated degradation.14 pVHL also acts on other HIF-independent pathways and inhibits both NF-kB and cyclin A activity.11 Through HIF-independent pathways, pVHL inhibits the production of many cytokines and growth factors.15 It also preserves chromosome stability,16 promotes cilia production,17 and acts on stabilization of RNA polymerase II subunit 1.18 The HIF transcription factor contains two subunits: the oxygen-sensitive a subunits (HIF1a, HIF2a and HIF3a) and the continually transcribed HIF1 subunit (also called the aryl hydrocarbon nuclear translocator (ARNT)).19 The interaction between pVHL and HIFa necessitates hydroxylation on either of two prolyl residues by members of the egl nine homolog (EglN) family.20 In order to be active, these enzymes need molecular oxygen, Fe (II), and 2-oxoglutarate. Under normal oxygen levels, the HIFa subunits are prolyl

FIGURE 1. Location of the VHL gene. The VHL tumor suppressor gene is located on the short arm of chromosome 3 (3p25-26). Genetic Home Reference. Source: http://ghr.nlm.nih.gov/gene/VHL. Seminars in Ophthalmology

Von Hippel-Lindau Disease 379 hydroxylated, ubiquitylated, then destroyed. During hypoxia, the HIFa subunit is not prolyl hydroxylated and is not recognized by pVHL and consequently not destroyed. It then heterodimerizes with HIF1 . The heterodimers enter the nucleus, recruit transcriptional co-activator complexes,21 and control the expression of target genes by binding to the hypoxia-response element.11 By activating HIF, the metabolism shifts to anaerobic glycolysis. It also stimulates secretion of proangiogenesis factors that include vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF- ), erythropoietin (Epo), and transforming growth factor (TGF- a), affect the remodeling of the extracellular matrix, and increase resistance to apoptosis and mobility.20 VHL-defective tumors are found to activate angiogenesis and the HIF pathway as reflected in siRNA inhibition of FGF reducing the proliferation of pulmonary artery smooth muscle cells.11

VHL Gene Mutations In VHL-associated tumors, an allelic deletion within the VHL gene locus has been described. Conforming to the ‘‘Knudson two-hit’’ model, one allele is first constitutively inactivated and carried in all of the cells and the other allele is inactivated later (second hit) at the somatic level. Consequently, it is thought that the pathology is associated to the somatic inactivation or loss of the remaining wild-type VHL allele.18,22 Malignant tumors in multiple organs result from this genetic inactivation, which initiates neoplastic growth. Sporadic cases would be due to the somatic biallelic inactivating VHL mutations. More than 900 different mutations of the VHL gene have been identified.23 In 2010, a study in the Netherlands24 analyzed mutations in 945 VHL families and found VHL type 1 families to have 43% missense mutations, 17% frameshift mutations, 13% nonsense, 9% splice, 8% inframe deletion/insertions, and 10% partial/complete deletions. Partial large gene deletions result in complete defect of protein function.11,23 In contrast, VHL type 2 is more likely associated with missense mutations affecting the protein-binding sites of the VHL protein.11,23 This mutation was also found in the Netherlands family study where missense mutations were found in 83.5% of VHL type 2 families.24 There also were a few cases with nonsense, frameshift, splice, in-frame deletion/ insertions, and partial deletions. VHL-associated ocular lesions include retinal and optic nerve head hemangioblastomas, and belong to types 1, 2A, and 2B.11 To date, only a few mutations upstream of the internal start codon 54 have been described (codons 25, 38, 46, and 52), and these mutations have been linked either with pheochromocytomas (codons 25 and 38) or with VHL !

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disease (codons E46X and E52K).23 Germline VHL gene mutations have been described in about 40% of familial or bilateral pheochromocytomas.1,25 Screening for germline VHL gene mutations may be suggested in patients even with apparently sporadic unilateral pheochromocytomas (ASP), as these cases are rarely due to sporadic VHL mutation25 and a germline mutation could be found in 25%.24 It is important to identify germline VHL gene mutations in these patients, as the onset of the VHL disease may show high variability and many patients with VHLrelated pheochromocytomas might have a higher risk for developing other manifestations of the disease.26

Perspective on VHL Tumorigenesis Recent studies suggest that VHL-associated neoplasms may not be caused by the sole mutation of the pVHL. Mutations were found to affect the HIFindependent pathways27 or other oncogenes or tumor suppressor genes.28 Mack et al. found that tumor growth was not promoted by the loss of the VHL gene.29 In contrast, in RCC the loss of the VHL gene promoted growth and the re-introduction of the wild-type VHL gene was able to suppress tumor growth.30,31 This finding suggests that additional mutations in RCC could account for the growth advantage in the setting of VHL deficiency. Studies on cytogenetic patterns of chromosomal loss and gain in human CCRCC (clear cell renal cell carcinoma) tumor tissue samples32 showed that the 3p loss is often associated with 5q gain due to unbalanced translocations, but this loss is usually followed by continual deletions on 3p due to genome instability.33,34 Patients with Class 2C VHL represent an example of pVHL tumorigenesis related to dysregulation of the HIF-independent pathway, since they still developed pheochromocytoma despite having a functioning VHL-HIF axis.8 Other findings supporting this hypothesis include the accumulation of JUNB (inhibitor of the pro-apoptotic molecule JUN) in pheochromocytoma, formation of renal and genital tract cysts from microtubule instability, and the dysregulation of Wnt signaling by preventing -catenin from degradation in CCRCC.32 Recently, an additional function of pVHL appears to be the marking of Skp2 for degradation in an E3 ubiquitin ligase-independent manner, resulting in increased p27/kip1, which results in S-phase arrest, thus preventing cell proliferation when there is DNA damage. In pVHL-deficient RCC tissue, Skp2 levels are found to be abnormally elevated with low p27/kip1.35 Also, it is hypothesized that there are unidentified neighboring genes in chromosome 3p that may have a tumorigenic function. Large deletion of these genes may be protective from tumor formation while point mutations would not affect these genes.36 For example, Brk1 maps near

380 N. M. N. Haddad et al. the VHL gene and acts as a regulator of the actin cytoskeleton. Loss of this gene is protective against tumors by inducing defects in cell migration in RCC and other tumors.36 This mapping might also explain the genotype-phenotype correlation in VHL patients with retinal hemangioblastomas.

VHL Genetic Screening Mutation analysis in VHL is highly specific and sensitive, making genetic testing more accurate in atrisk relatives, even if the proband cannot be tested, and when these relatives carry de-novo mutations.24,38 There are numerous recommendations for VHL screening, including individuals with at least two of the following VHL manifestations: neuroendocrine tumors, paragangliomas, familial history of renal cancers, endolymphatic sac tumors, hemangioblastomas, cysts of the pancreas and/or pheochromocytomas.37 Screening has also been suggested if a person has one VHL-affected first-degree relative and exhibits at least one of the mentioned manifestations.6 Mutation analysis is recommended in the presence of a VHL germline mutation in the family or when there is a positive family history of pheochromocytomas, hemangioblastomas or RCC.24 Hes et al.39 recommend genetic screening in the presence of VHL-associated tumors (hemangioblastomas, pheochromocytomas) in young patients (younger than 50 years), or RCC in patients younger than 30 years. These screening recommendations are related to the clinical diagnosis of VHL. Clinical criteria for positive diagnosis are defined as either more than one central nervous system (CNS) or retinal hemangioblastoma, either a single CNS or retinal hemangioblastoma associated with visceral complications (multiple pancreatic, renal or hepatic cysts; pheochromocytoma; renal cancer) with the exception of epididymal cysts, or any of the previously mentioned pathologies with a positive family history.40–42 As noted above, germline mutations are also suggested in the cases of ASP and in apparent sporadic retinal43 or CNS hemangioblastomas.44 Presymptomatic testing for VHL is usually offered after appropriate genetic counseling about the implications of this condition. Therefore, scanning the genome presents a number of challenges for the genetic counselor. Further understanding of the psychosocial effects of VHL screening is needed for appropriate pre- and post-test counseling.45

Evaluation of Patients Diagnosed with VHL Disease Management of VHL requires appropriate general screening, early detection and management of the

tumors, and exploration of reproductive options when needed. The initial presentation of VHL patients is varied. A comprehensive evaluation of patients should include a clinical history, physical examination, and laboratory and imaging evaluations to identify the different manifestations of the disease. Imaging evaluations may include abdominal ultrasonography yearly, starting at age eight years,46 computed tomography, and brain and spinal cord magnetic resonance imaging starting at age 11 years, and then every two years with emphasis on the posterior fossa. Dilated retinal fundus examination should start early in infancy and be continued yearly.46 Yearly biochemical and laboratory tests starting at age two years or in the presence of high blood pressure40,41,46 should include routine biochemical testing and 24-hour urinary catecholamine metabolite determination.23,20

Ocular Findings in VHL Syndrome Retinal hemangioblastomas are the most common and earliest presentation of VHL disease, generally in patients in their mid-twenties. In Chew’s series, ocular disease was found in 205 of the 406 patients with VHL disease.10 Patients presenting with isolated retinal hemangioblastomas have a 30% risk of developing VHL disease.44 Lesions are usually located in the retinal periphery, most commonly in the supero temporal quadrant, are often multiple and bilateral, are red-colored, highly vascular with a globular shape, and vary in size from 10 microns up to 3000 microns or more (Figures 2 and 3).43 The lesions may be on or within one disc diameter of the optic nerve head in up to 15% of the cases. Vitreous and retinal hemorrhages are uncommon. The diagnosis is generally made by

FIGURE 2. Retinal hemangioblastoma found in the retinal periphery showing globular shaped, red-colored and highly vascular tumor. Photograph courtesy of Robert W. Cavicchi, CRA, FOPS. Seminars in Ophthalmology

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FIGURE 5. Same retinal heamgioblastoma as in Figure 2 after laser photocoagulation treatment. Photograph courtesy of Robert W. Cavicchi, CRA, FOPS.

FIGURE 3. Retinal Hemangioblastoma: Large globular tumor with dilated feeder vessel arising from the optic nerve. Note also the exudation and macular edema. Photograph courtesy of Paolo Silva, MD.

FIGURE 6. Same retinal hemangioblastoma as in Figures 2 and 5, ten years after laser photocoagulation treatment. Photograph courtesy of Robert W. Cavicchi, CRA, FOPS.

FIGURE 4. Ultrawide field photograph showing treated lesions with laser photocoagulation above the optic disc and peripheral superior temporal and inferior fields. Photograph courtesy of Robert W. Cavicchi, CRA, FOPS.

funduscopy, where the tumor and its feeder vessels are seen along with exudation and subretinal fluid. However, with the increasing availability of ultrawide field imaging, peripheral tumors can be more readily and accurately documented and monitored over time47 (Figure 4). Fluorescein angiography (FA) may aid in differential diagnosis or in planning treatment due to a generally distinctive presentation. The feeder vessel to the tumor will hyperfluoresce during the arterial phase of the FA and the draining vein is prominent during the venous phase. The tumor itself !

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exhibits hyperfluorescence and will demonstrate leakage of dye in the later phases of the FA. Ultrawide field angiography may facilitate the detection of small peripheral angiomas that would have been difficult to detect on funduscopy and which are the most amenable to treatment. Differential diagnoses of VHL-associated retinal hemangioblastomas include racemose hemangioma (Wyburn-Mason disease), Coats’ disease, retinal macroaneurysm, retinal cavernous hemangioma, and vasoproliferative retinal tumor.48 Current treatment for retinal hemangioblastomas is based on the principle of tumor ablation and includes laser photocoagulation (Figures 5 and 6), cryotherapy, radiation, photodynamic therapy, or a combination of modalities. Rarely, surgical excision may be performed for severe cases, but a high rate of recurrences and complications has been reported.49 The primary goal of treatment is to prevent further growth of the

382 N. M. N. Haddad et al. lesions, which can lead to vision loss due to tractional retinal detachment, exudation, hemorrhage, glaucoma, and cataract.10,50 Well-timed early treatments are effective in preserving vision and preventing blindness. In a large series of 406 patients (199 families) with ocular VHL disease, visual acuity was 20/200 or worse in approximately 8.4% of the eyes and enucleation had to be performed in 8.1% of these eyes,11 emphasizing the potential for visual complications and the need for close monitoring of patients at risk. Individuals with VHL need to have their retinas evaluated at least annually.51 Patients with more severe disease should be followed more frequently.

Physiopathology and Histopathology of Ocular Hemangioblastomas in VHL Disease As noted above, mutations in pVHL result in a lower breakdown of HIF, leading to a higher secretion of pro angiogenic factors and therefore the development of retinal hemangioblastomas. pVHL can also induce tumorigenesis through non–HIF-mediated pathways, including cell–extracellular matrix interactions and cell– cell adhesion, which also can lead to retinal hemangioblastomas formation.11

Retinal Hemangioblastomas Retinal and optic nerve head hemangioblastomas share histopathological similarities with CNS hemangioblastomas. The tumors are composed of abnormal capillary-like fenestrated channels bordered by vacuolated foamy cells. The tumors substitute the entire layers of the neuroretina.11 The tumors are known to lack endothelial cells and some have glial cell proliferation, usually at the margins of relatively large hemangioblastomas. The vacuolated stromal cells were found to have loss of heterozygosity (LOH) of the VHL gene. This feature was not found in the vascular endothelial or reactive glial cells of 15 retinal and 4 optic nerve head hemangioblastomas analyzed by Chan et al.11 Thus, these vacuolated foamy ‘‘stromal’’ cells may derive from the arrested hemangioblast progenitor and contain a deletion of one VHL gene wild-type allele, supporting the idea that these are the ‘‘true tumor cells’’ of the disease which affect the adjacent endothelial cells.

Optic Nerve Head Capillary Hemangioblastomas Optic nerve head capillary or juxtapapillary capillary hemangioblastomas are associated with VHL disease. Given their location next to the optic nerve head, they

are hard to treat and therefore visual prognosis is guarded.52 The tumors present initially as small lesions at the optic disc or in the peripapillary region. They have an indolent course, increase in size very slowly over a number of years, but with increased growth and vascular leakage visual acuity worsens due to retinal edema, epiretinal membranes, hard exudates and, in extreme cases, detachment of the macula.53 As noted above, juxtapapillary and optic nerve head hemangioblastomas cannot be treated by laser or other surgical ablative techniques the same way peripheral retinal hemangioblastomas are treated because such treatment can result in substantial loss of vision.54 Optic nerve head lesions present a treatment challenge because, if left untreated, their progression ultimately can lead to total blindness and loss of the eye.55 Differential diagnoses of optic nerve head hemangioblastoma include papillitis, disk edema, anterior ischemic optic neuropathy, and sarcoidosis.55

Factors Influencing Hemangioblastoma Progression Toy et al.56 reported the following factors to be significantly linked to anatomic progression of retinal hemangioblastomas: younger age at onset of ocular VHL disease, younger age at baseline visit, and involvement of the fellow eye with ocular VHL disease. They did not find a correlation between the presence of extraocular VHL disease and anatomic progression of ocular VHL disease. They worked on genotype-phenotype correlations and found that the genotype category of the germline VHL mutation was associated with ocular VHL disease progression using Kaplan-Meier time-to-event analyses on the basis of individual participants, even if not significantly associated with disease progression in univariate or multivariate analyses.56 For example, complete deletion mutation of the VHL gene was protective and development of retinal hemangioblastomas in these patients was significantly lower relative to patients with a missense or protein-truncating mutation. Moreover, those with complete deletions have better visual acuity and decreased incidence of retinal hemangioblastomas. Missense mutations resulted in a higher incidence of ocular disease and juxtapapillary lesions.43,57,58

Hemangioblastomas and Their Relationship to VEGF In the absence of pVHL, all ocular hemangioblastomas were found to express high levels of VEGF, HIF, ubiquitin, Epo, and EpoR.59 VEGF was detected in the neovasculature of fibrovascular Seminars in Ophthalmology

Von Hippel-Lindau Disease 383 epiretinal membranes, and elevated ocular levels have been demonstrated in patients with VHL disease.60 In addition, central nervous system hemangioblastomas expressed an upregulation of VEGF receptors.61 VEGF, ubiquitin, HIF, and Epo are known to stimulate angiogenesis and hypervascularization. Recent studies also suggested that progression of VHL-related tumors is dependent on vascular endothelial growth factor (VEGF).62,63 VEGF is a homodimeric glycoprotein cytokine originally identified for its effects on endothelial cell proliferation and vascular permeability.64–67 VEGF receptors on endothelial cells are part of the tyrosine kinase family of receptors. Two principle receptors have been identified: Flt-1 and KDR (or VEGFR-1 and 2, respectively), through which VEGF induces physiologic and pathophysiologic angiogenesis.68 Overproduction of VEGF in the mouse brain causes lesions that are similar to hemangioblastomas.69 Clearly, the highly vascular nature of VHLrelated tumors might be explained by the increased expression of VEGF; therefore, VEGF suppression might represent a target for systemic therapy. Many VEGF-targeted strategies have been tested in clinical trials, including anti-VEGF antibodies and small molecule inhibitors of VEGFR-1 and 2.70–72 For example, SU5416, the small molecule VEGFR inhibitor, has been tested73 and Aiello et al.67 reported its use in a case of VHL syndrome and optic nerve head hemangioblastoma. The individual had lost one eye due to retinal angiomatosis, and the remaining eye had reduction in visual acuity, contrast sensitivity, and visual field as a result of worsening optic nerve head hemangioblastoma. After systemic treatment with SU5416, there was a rapid improvement of all visual function parameters, with all measures normalizing within one month of initiating therapy, with an improvement remaining essentially stable for 18 months. Although the lesion stopped growing in size, there was no observable reduction in the dimensions of the optic nerve head lesion on fundus examination. It was hypothesized by the authors that either edema in or around the optic disc or a nonvisualized component of the hemangioblastoma was affected.67 Other molecules, like many stem cell markers, including CD133, Epo, and EpoR, have also been found in ocular hemangioblastomas associated with VHL.74 CD133 is a surface molecule expressed on progenitors from hematopoietic, endothelial, and neural lineages. Hemangioblastomas were found to comprise CD133 positive cells in different amounts.74 Epo and EpoR have been identified in various VHL-associated lesions, including CNS hemangioblastomas.75 However, in ocular VHL-associated hemangioblastomas, EpoR is more expressed than Epo.11 Furthermore, through the HIF pathway, there is an increase in the secretion of cytokines and growth factors such as TGF, FGF, PDGF, and EGF.56 !

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In addition, Chan et al. detected expression of CXCR4 in VHL-associated retinal hemangioblastomas. CXCR4 is a chemokine receptor. HIF-induced product involved in the migration of hematopoietic progenitors and stem cells, which, under normoxic conditions, is downregulated by pVHL, and in hypoxic conditions, attracts and regulates the migration and development of embryonic VHL cells in the eye.11 Treatment targeting specific growth factors associated with VHL disease is a promising approach for lesions at or close to the optic nerve head.

Novel Therapeutic Approaches In the presence of multiple lesions or lesions at or near the optic nerve head which are not readily amenable to treatment by ablative techniques, the elucidation of the physiopathology of VHL disease has provided novel therapeutic targets. Inhibition of growth factors such as VEGF or PDGF67,76 or inhibitors of the numerous different steps in the pathway of pVHL, such as cycline-depending kinase blockers, are all potential therapies. In early reports, findings after such inhibition have suggested that, despite improvements in visual acuity and decrease in retinal exudates and edema,77 there has been no observable anatomical regression of hemangioblastomas themselves.67,76,78 The efficacy of anti-angiogenesis molecules such as SU5416, bevacizumab, ranibizumab, sunitinib, and pegaptanib as single agents seems to be inadequate. The development of novel therapeutic strategies for ocular VHL may necessitate targeting the real tumor cells directly. Inhibition of CXCR4, Epo/EpoR, and other molecules involved in the pVHL pathway could also be a therapeutic option. Other studies are focusing on stem cell transplantation and tumor vaccine as other approaches for the treatment of VHL disease.79 Also, drugs such as topotecan and digoxin can successfully block HIF synthesis at low concentrations33 and might prove beneficial for optic hemangioblastomas in which current treatment modalities have limited efficacy.

SUMMARY AND CONCLUSIONS Studying the genetics of VHL has increased our understanding of the physiopathology of the disease. Analyzing HIF pathways and HIF-independent pathways identifies new therapeutic targets. Genetic screening and diagnosis can help identify patients at risk and provide for earlier intervention with decreased morbidity from the disease. Genotypephenotype correlation studies have demonstrated that patients with missense and protein-truncating mutation are at increased risk of developing retinal

384 N. M. N. Haddad et al. hemangioblastomas. These sorts of pathophysiologic and genetic advances have laid the foundation for future approaches that may provide improved prediction of onset or progression of pathologic lesions and the eventual development of less-invasive, individualized, targeted novel therapies.

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DECLARATION OF INTEREST 15.

The authors declare that there are no conflicts of interest. The authors alone are responsible for the writing and content of the paper.

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