Sporadic ccRCC Research Articles

Characterization of VHL missense mutations in sporadic clear cell renal cell carcinoma: hotspots, affected binding domains, functional impact on pVHL and therapeutic relevance.

BackgroundThe VHL protein (pVHL) is a multiadaptor protein that interacts with more than 30 different binding partners involved in many oncogenic processes. About 70 % of clear cell renal cell carcinoma (ccRCC) have VHL mutations with varying impact on pVHL function. Loss of pVHL function leads to the accumulation of Hypoxia Inducible Factor (HIF), which is targeted by current targeted treatments. In contrast to nonsense and frameshift mutations that highly likely nullify pVHL multipurpose functions, missense mutations may rather specifically influence the binding capability of pVHL to its partners. The affected pathways may offer predictive clues to therapy and response to treatment. In this study we focused on the VHL missense mutation pattern in ccRCC, and studied their potential effects on pVHL protein stability and binding partners and discussed treatment options.MethodsWe sequenced VHL in 360 sporadic ccRCC FFPE samples and compared observed and expected frequency of missense mutations in 32 different binding domains. The prediction of the impact of those mutations on protein stability and function was assessed in silico. The response to HIF-related, anti-angiogenic treatment of 30 patients with known VHL mutation status was also investigated.ResultsWe identified 254 VHL mutations (68.3 % of the cases) including 89 missense mutations (35 %). Codons Ser65, Asn78, Ser80, Trp117 and Leu184 represented hotspots and missense mutations in Trp117 and Leu 184 were predicted to highly destabilize pVHL. About 40 % of VHL missense mutations were predicted to cause severe protein malfunction. The pVHL binding domains for HIF1AN, BCL2L11, HIF1/2α, RPB1, PRKCZ, aPKC-λ/ι, EEF1A1, CCT-ζ-2, and Cullin2 were preferentially affected. These binding partners are mainly acting in transcriptional regulation, apoptosis and ubiquitin ligation. There was no correlation between VHL mutation status and response to treatment.ConclusionsVHL missense mutations may exert mild, moderate or strong impact on pVHL stability. Besides the HIF binding domain, other pVHL binding sites seem to be non-randomly altered by missense mutations. In contrast to LOF mutations that affect all the different pathways normally controlled by pVHL, missense mutations may be rather appropriate for designing tailor-made treatment strategies for ccRCC.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2688-0) contains supplementary material, which is available to authorized users.

Loss ofvhlin the zebrafish pronephros recapitulates early stages of human clear cell renal cell carcinoma

ABSTRACTPatients with von Hippel–Lindau (VHL) disease harbor a germline mutation in the VHL gene leading to the development of several tumor types including clear cell renal cell carcinoma (ccRCC). In addition, the VHL gene is inactivated in over 90% of sporadic ccRCC cases. ‘Clear cell’ tumors contain large, proliferating cells with ‘clear cytoplasm’, and a reduced number of cilia. VHL inactivation leads to the stabilization of hypoxia inducible factors 1a and 2a [HIF1a and HIF2a (HIF2a is also known as EPAS1)] with consequent up-regulation of specific target genes involved in cell proliferation, angiogenesis and erythropoiesis. A zebrafish model with a homozygous inactivation in the VHL gene (vhl−/−) recapitulates several aspects of the human disease, including development of highly vascular lesions in the brain and the retina and erythrocytosis. Here, we characterize for the first time the epithelial abnormalities present in the kidney of the vhl−/− zebrafish larvae as a first step in building a model of ccRCC in zebrafish. Our data show that the vhl−/− zebrafish kidney is characterized by an increased tubule diameter, disorganized cilia, the dramatic formation of cytoplasmic lipid vesicles, glycogen accumulation, aberrant cell proliferation and abnormal apoptosis. This phenotype of the vhl−/− pronephros is reminiscent of clear cell histology, indicating that the vhl−/− mutant zebrafish might serve as a model of early stage RCC. Treatment of vhl−/− zebrafish embryos with a small-molecule HIF2a inhibitor rescued the pronephric abnormalities, underscoring the value of the zebrafish model in drug discovery for treatment of VHL disease and ccRCC.

Key considerations in the treatment of von Hippel-Lindau disease.

10.2217/fon-2016-0028 © 2016 Future Medicine Ltd Cancer initiation and progression is the result of an accumulation of mutations in key tumor suppressor genes, mismatch repair genes and oncogenes which alter cancer cell growth, death and differentiation. Mutations occurring in cancer tissue are termed somatic; whereas, heritable mutations that may be passed on to subsequent generations occur in germline DNA. Deleterious germline mutations in certain genes can lead to hereditary cancer syndromes whereby family members carrying the mutation have an increased susceptibility to certain tumor phenotypes. Germline mutations in the von HippelLindau (VHL) gene, a tumor suppressor found on chromosome 3p25, are inherited in an autosomal dominant fashion giving way to the potential development of a spectrum of tumor types including: renal and pancreatic cysts, clear cell renal cell carcinoma (ccRCC), hemangioblastomas (HB), pheochromocytomas, retinal angiomas and pancreatic neuroendocrine tumors (PNETs) [1,2]. VHL disease occurs in approximately one in 35,000 births, and the morbidity and mortality associated with VHL disease primarily centers around the neurologic complications of HBs and the progression of ccRCC [3,4]. VHL lesions, despite different anatomic origins, are in general highly vascular owing to the loss of the underlying anti-angiogenic function of the VHL gene. Given the variety of tumor types within a single individual with VHL disease, treatment necessitates a personalized, multidisciplinary approach; and, given that the most frequent alterations in sporadic ccRCC involve loss of the 3p chromosomal arm including the VHL gene, treatment discoveries for this rare, heritable disease have implications for a much wider patient population [5]. In this article, we discuss the current state of treatment options for VHL disease across anatomic sites of expression in the context of its molecular underpinnings. COMMENTARY

Abstract 1375: Epigenetic changes associated with resistance to sunitinib in human clear cell renal cell carcinoma models

Abstract Background: Sunitinib is first-line therapy for patients with advanced clear cell renal cell carcinoma (ccRCC). Despite the clinical efficacy of sunitinib, in the majority of patients the disease eventually develops resistance and progresses. Epigenetic modifications of histone protein in chromatins have been shown to play a role in the regulation of gene transcription patterns in cells primarily by the catalytic activity of histone methyltransferase. EZH2 has been shown to contribute to tumor angiogenesis by inactivating anti-angiogenic factors via methylation at their promoter region. In this study, we examined epigenetic changes that may be associated with response and the resistant phenotype. Methods: Human ccRCC xenograft models RP-01 (isolated from a skin metastasis in a patient with sporadic ccRCC) and RP-02 (isolated from a skin metastasis in a patient with hereditary ccRCC) were implanted (∼1mm pieces) subcutaneously into SCID mice and were randomly assigned into two groups (sunitinib and vehicle). Mice were treated 5 days/week with sunitinib at 40mg/kg. Tumor volumes and body weights were assessed weekly by caliper measurements and weigh scale, respectively. Tumor tissues and blood were collected prior treatments and when tumors became resistant to treatment. Tissues collected were used for immunohistochemistry studies and RNA analysis. Results: Our results showed that RP-01 and RP-02 tumors, although initially responsive to treatment at 40 mg/kg, eventually became resistant to treatment. Immunohistochemistry analysis of RP-01 tumor samples indicated tumors to be hypo-vasculature when responding to sunitinib, but then became hyper-vascularized at point of resistance by CD31 staining. Analysis also revealed an associated increase in the expression of the methyltransferase EZH2 when tumors became resistant to sunitinib. However, there was no change in the expression levels of its EZH2 associated histone make H3K27me3. In addition, we noticed an increase in the levels of H3k9me2 and H3k4me2 in tumors resistant to sunitinib as compared to controls. Conclusion: Overall, our results suggest the potential role of epigenetic changes that that may be associated with sunitinib-induced resistance in ccRCC Citation Format: REMI ADELAIYE, Kiersten Marie Miles, Eric Ciamporcero, Dylan Conroy, Swathi Ramakrishnan, Ashley Orillion, Sheng Yu Ku, May Elbanna, Li shen, Sreenivasulu Chintala, Roberto Pili. Epigenetic changes associated with resistance to sunitinib in human clear cell renal cell carcinoma models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1375. doi:10.1158/1538-7445.AM2014-1375

New Insights into von Hippel-Lindau Function Highlighted by Investigation of the Trichloroethylene-Induced p.P81S Hotspot Mutation

The von Hippel-Lindau gene (VHL), composed of three exons, encodes a protein, pVHL, with diverse tumor suppressor activities. Germline VHL mutations are found in patients with von HippelLindau (VHL), an autosomal dominant hereditary cancer syndrome in which affected individuals are at risk for the development of highly vascularized benign and malignant tumors including central nervous system hemangioblastomas, retinal hemangiomas, clear cell renal carcinomas (ccRCCs), pheochromocytomas, and pancreatic neuroendocrine tumors (1). Biallelic inactivation of VHL, whether via mutation or promoter methylation, is found in VHL-associated tumors and in nearly 90% of sporadic ccRCC tumors (2,3), the most common form of sporadic kidney cancer. Among its activities, pVHL functions as the substrate recognition module of a multiprotein ubiquitin ligase complex, and, by recruiting hypoxia-inducible factor (HIF) 1α and 2α to this complex, mediates their oxygen-dependent degradation (4). VHL mutations that interfere with HIF-α recognition promote aerobic glycolysis and support neoangiogenesis, perhaps the most intensively studied consequences of pVHL loss. However, regulation of HIF-α expression is unlikely to be the sole mechanism underlying the tumor suppressor function of pVHL. Given its role as the substrate recognition module of a multiprotein complex, it is reasonable to assume that pVHL has additional activities that depend on protein–protein interactions unrelated to (or perhaps complementary with) HIF stabilization, and that deregulation of one or more of these activities contributes to tumor formation, likely in a tissue-specific manner. Indeed, pVHL has been implicated in a number of cellular processes that appear to be HIF independent, including cell cycle regulation, extracellular matrix assembly, and cytoskeleton stability (5). The development of ccRCC is associated with both genetic (eg, VHL germline mutation) and environmental factors, particularly long-term exposure to the industrial solvent trichloroethylene (TCE). Although TCE exposure does not affect the histopathology of sporadic ccRCC, its onset is earlier in TCE-exposed individuals, and the VHL gene in a substantial percentage of these tumors contains a specific mutation of a cytosine to thymidine at nucleo

PBRM1 and BAP1 as Novel Targets for Renal Cell Carcinoma

Technological advances in genome sequencing have led to the identification of novel driver genes mutated in renal cancer. Hitherto, 1 gene was known to be frequently mutated in renal cell carcinoma of clear cell type (ccRCC), the von Hippel-Lindau (VHL) gene. VHL was identified by positional cloning as the gene responsible for a familial syndrome with renal cancer predisposition, von Hippel-Lindau. Subsequently, VHL was found to be inactivated in approximately 90% of sporadic ccRCC. The discovery of VHL, together with the elucidation of its function, transformed the treatment of ccRCC leading to the introduction of 5 new drugs into the clinic. However, no other familial ccRCC predisposing genes are frequently mutated in sporadic ccRCC. With the development of massively parallel sequencing, a plethora of somatically mutated genes has been identified. Most genes are mutated at low frequencies, but 3 genes are mutated in more than 10% of ccRCC, PBRM1 (mutated in ~50%), BAP1 (~15%), and SETD2 (~15%). Like VHL, all 3 genes are 2-hit tumor suppressor genes. Furthermore, these 3 genes are within a 50-Mb region on the short arm of chromosome 3p that encompasses VHL and is deleted in ~90% of ccRCC. We discovered that PBRM1 mutations tend to anticorrelate with BAP1 mutations in ccRCC and that PBRM1- and BAP1-mutated tumors exhibit different biology and are associated with markedly different outcomes. This established the foundation for the first molecular genetic classification of sporadic ccRCC. Herein, I review the evidence that implicated PBRM1 and BAP1 as renal cancer driver genes, provide an update on the function of the gene products, and speculate on how mutations in these genes may be exploited therapeutically.

Abstract A18: The role of histone demethylase JARID1C in tumorigenesis of clear cell renal cell carcinoma

Abstract Clear cell renal cell carcinoma (ccRCC) constitutes 75% of all RCC. It is characterized by inactivation of the tumor suppressor Von Hippel Lindau gene (VHL) in ~70% of sporadic ccRCC cases. Recent large scale screen of ~3,500 genes by PCR-based exon re-sequencing identified several new cancer genes in ccRCC including UTX, JARID1C and SETD2. These genes encode enzymes that demethylate and methylate key lysine residue of histone H3. In particular, JARID1C is a histone 3 lysine 4 (H3K4) demethylase involved in the demethylation of gene activating trimethyl H3K4 (H3K4me3) mark and was found to be mutated in 3-9% of ccRCC, independent of the mutation status of VHL. Using gene expression profiling, we revealed that major down-regulated pathways in primary RCCs harboring JARID1C inactivating mutation are pointing towards changes involving metastatic properties. The highlighted pathways include ECM-receptor interaction, focal adhesion, regulation of actin cytoskeleton, cell junction organization and cell junction assembly. Therefore, we focused on elucidating the role of JARID1C in ccRCC invasive properties. From in vitro assay, depletion of JARID1C by introducing JARID1C shRNA in ccRCC cells did not result in significant changes in cell migration. By contrast, in consistent with the findings from gene expression profiling, we showed that such depletion of JARID1C significantly enhances the invasiveness of ccRCC cells regardless of their VHL gene status. In summary, our findings establish the role of JARID1C in potentiating the invasive capabilities of ccRCC. Citation Format: Lian Dee Ler, George Edward Allen, Swe Swe Myint, Bin Tean Teh. The role of histone demethylase JARID1C in tumorigenesis of clear cell renal cell carcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr A18.

Carbonic anhydrase IX (CAIX) is not an independent predictor of outcome in patients with clear cell renal cell carcinoma (ccRCC) after long‐term follow‐up

To evaluate carbonic anhydrase IX (CAIX) expression as an independent prognostic marker for clear cell renal cell carcinoma (ccRCC). With recent smaller studies showing conflicting results, we aimed to update our initial analysis in 2007 with an additional 5-year follow-up. We provided long-term follow-up of the same cohort used in our 2007 study (730 patients with unilateral, sporadic ccRCC treated surgically between 1990 and 1999). Associations of CAIX expression with RCC death and distant metastases were evaluated using Cox proportional hazards regression models. CAIX was expressed in 708 (97.0%) of the specimens; 163 tumours (22.3%) had low (≤85%) expression and 567 (77.7%) high (>85%) expression. There were 483 deaths and 265 RCC-specific deaths. The median follow-up for the 247 patients still under observation was 13.8 years. Univariately, low CAIX expression was associated with an increased risk of RCC death vs high expression (hazard ratio 1.62; P < 0.001). Low CAIX expression was not statistically significantly associated with RCC death or distant metastases after adjusting for nuclear grade or coagulative tumour necrosis. After additional long-term follow-up of our large cohort, our results continue to suggest that CAIX is not an independent prognostic marker for ccRCC.

Expression of CIDE proteins in clear cell renal cell carcinoma and their prognostic significance

Clear cell renal cell carcinoma (ccRCC) is the major and aggressive subtype of renal cell carcinoma. It is known to derive its histologic appearance from accumulation of abundant lipids and glycogens. The cell death-inducing DFF45-like effector (CIDE) family has been characterized as the lipid droplet proteins involved in the metabolism of lipid storage droplets. The purpose of this study was to evaluate the expression of CIDE proteins in ccRCC cells and to investigate their prognostic significance. We examined consecutive patients with sporadic ccRCC, who underwent nephrectomy, to measure their mRNA and protein expression of CIDE proteins. We found that Cidec and ADRP expression were significantly up-regulated in ccRCC, compared with normal kidney tissues. Cideb was down-regulated. We also found that Cideb was expressed more in low-grade ccRCC than in high-grade tumors. To further clarify the relationship between Cideb expression and patient prognosis, we evaluated 57 ccRCC patients followed up for 120 months. Reduced ccRCC Cideb expression was associated with a higher Fuhrman nuclear grade. Patients with high Cideb expression had better overall survival rate than those with low expression (p < 0.05). Cideb expression was an independent predictor of survival (p = 0.001). Although the biologic function of Cideb in ccRCC remains unknown, the expression level of Cideb might be a novel predictor of prognosis in ccRCC.Electronic supplementary materialThe online version of this article (doi:10.1007/s11010-013-1605-y) contains supplementary material, which is available to authorized users.

Hematopoietic Functions of the Tumor Supressor Von Hippel Lindau (VHL) in the Etiology of Hemangioblastoma.

AbstractAbstract 2425Germline heterozygous mutation in the VHL tumor suppressor gene is the underlying genetic defect in VHL disease. Cancer arises when the function of the remaining wildtype allele is lost. VHL patients are prone to develop renal cell carcinoma of the clear cell type (ccRCC) and hemangioblastoma (HB). VHL mutations underlie also the majority of sporadic ccRCC and HB. HB are highly vascularized tumors of the central nervous system. However, it is unclear from which tissue HB arise (reviewed in (1)). Although HB are sometimes described as vascular tumors, inactivation of both VHL alleles has been detected only in the stromal component, suggesting that abnormal angiogenesis is driven by cytokines released from the tumor rather than by VHL inactivation in the endothelium. There is evidence that hematopoietic stem cells or early progenitor cells are involved in the formation of HB. For instance, advanced HB are frequently accompanied by extramedullary erythropoiesis (EME) (2,3).Previously, overgrowth of blood vessels (hemangioma) has been observed in the liver of constitutive heterozygous Vhl knock-out mice, and in a hepatocyte specific Vhl conditional knock-out (albumin-cre) (4). Here, we present a mouse model that may shed light on another aspect of HB, e.g. EME. We used HOXB7-cre for the conditional deletion of VHL in a subset of kidney tubules (HOXB7-Vhl mice). Interestingly, we found that these mice developed not only a kidney phenotype, but also liver lesions. At 2–3 months of age, blood filled bulges developed close to the gall bladder (see Fig. 1A). Superficially, these lesions resembled previously described hemangiomas. However, histology revealed large blood lakunae that were not lined by blood vessels (Fig 1B). Hence, lesions appeared to arise not due to an overgrowth of blood vessels, but due to EME, with massive accumulation of red blood cells surrounding foci of nucleated progenitor cells (Fig. 1C). [Display omitted] CFU assays with liver cell suspensions confirmed EME: there was a significant increase in the number of liver BFUs in HOXB7-Vhl mice. HOXB7-Vhl mice had also elevated levels of EPO in the serum, and flow cytometry showed an increase in TER119+CD71+ erythrocyte progenitors in the spleen, but not the bone marrow. Despite EME and elevated EPO levels, HOXB7-VHL mice displayed only rarely polycythemia. In this context, it is noteworthy that VHL patients also present frequently with elevated EPO levels, without developing polycythemia. Interestingly, the majority of HoxB7-VHL mice developed thrombocytopenia, possibly due to the leakiness of the blood vasculature in the liver lesions. However, flow cytometry showed also a decrease in megakaryocytes (CD41+) in both bone marrow and spleen, and in addition to an increase of erythrocyte progenitors, an increase in TER119+CD41+ common megakaryocyte-erythrocyte progenitors (MEP) in the spleen. It is therefore possible that differentiation of the MEP is shifted towards the erythrocyte lineage in the HOXB7-Vhl mouse.We found no evidence of Vhl deletion in the liver of HOXB7-Vhl mice using LacZ reporter mice. Our mouse model suggests therefore a systemic etiology of HB. One of the best known functions of VHL is the negative regulation of the hypoxia-inducible factor (HIF). Besides EPO, the chemokine receptor CXCR4 is also a known HIF-responsive gene (5) that could be potentially disregulated by Vhl deletion. Furthermore, preliminary data indicate that constitutive heterozygous VHL mice show 50% mortality in transplantation experiments, irrespective of the genotype of transplanted stem cells (e.g. wt or VHL +/−). Hence, in addition to cell autonomous and systemic mechanisms, defects in the bone marrow environment may underlie hematologic presentations of VHL deficiency. Disclosures:No relevant conflicts of interest to declare.

Abstract 2329: Anti-tumor efficacy of Dll4 blockade in combination with sunitinib or aflibercept in primary human renal cell carcinoma models

Abstract Background: The Notch ligand Delta-like 4 (Dll4) is highly expressed in vascular endothelium and has been shown to play a pivotal role in regulating tumor angiogenesis, particularly angiogenic sprouting. Blockade of the Dll4-Notch pathway leads to an excessive production of aberrant non-functional tumor vascular structures and is associated with reduced tumor growth. REGN1035, an antibody that selectively binds and blocks murine Dll4, has demonstrated potent inhibition of tumor growth in a broad spectrum of xenograft models. The aim of this study was to assess the effect of REGN1035 alone and in combination with anti-VEGF therapy in two preclinical models of primary RCC. We hypothesized that combining REGN1035 with sunitinib or the VEGF blocker aflibercept would provide greater anti-tumor and anti-angiogenesis efficacy compared to single agent treatments. Methods: Two human RCC models isolated from skin metastases from a patient with sporadic ccRCC (RP-01) and a patient with hereditary ccRCC (RP-02), respectively, were bilaterally implanted subcutaneously into the flank area of three sets of 32 SCID mice (8 mice/group). When tumor volume reached ∼ 25 mm2, mice were treated with vehicle (human Fc control protein, 4.89 mg/kg, 1x/wk, s.q.), REGN1035 (5 mg/kg, 1x/wk, s.q.), sunitinib (40 mg/kg, 5x/wk, oral gavage), aflibercept (10 mg/kg, 2x/wk, s.q.) or REGN1035 plus sunitinib or aflibercept. Tumor growth was assessed with serial caliper measurements. Once tumor volume of vehicle mice reached 200 mm2, mice were sacrificed and tumor tissue was excised and analyzed. Results: In the RP-01 and RP-02 models, sunitinib and REGN1035 induced 60-69% and 71-81% inhibition (p &amp;lt; .01 vs. control), respectively, while the combination group showed 89-114% inhibition (p &amp;lt; .001 vs. single agents), respectively. No overt signs of toxicity (i.e., weight loss, diarrhea) were observed in any of the experimental groups. Similar results were observed by combining the VEGF blocker aflibercept with REGN1035. HIF-1α and HIF-2α protein levels in the tumor were both found to be downregulated in the combination treated mice, as compared to the single agent treated mice as monitored by Western blotting analysis. A reduction in microvessel density, as measured by CD31 and VEGF immunohistochemistry, was observed in the sunitinib treated group, whereas an increase in abnormal vascular structures was seen in the REGN1035 group. Summary: Overall, the data demonstrate that combination therapy with REGN1035 and sunitinib or aflibercept resulted in significant anti-tumor efficacy including tumor regression in primary human RCC xenografts and highlights the potential therapeutic application of combined VEGF and Dll4 blockade for the treatment of RCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2329. doi:1538-7445.AM2012-2329

Integrative Genomic Analyses of Sporadic Clear Cell Renal Cell Carcinoma Define Disease Subtypes and Potential New Therapeutic Targets

Sporadic clear cell renal cell carcinoma (ccRCC), the most common type of adult kidney cancer, is often associated with genomic copy number aberrations on chromosomes 3p and 5q. Aberrations on chromosome 3p are associated with inactivation of the tumor suppressor gene von-Hippel Lindau (VHL), which activates the hypoxia-inducible factors HIF1α and HIF2α. In contrast, ccRCC genes on chromosome 5q remain to be defined. In this study, we conducted an integrated analysis of high-density copy number and gene expression data for 54 sporadic ccRCC tumors that identified the secreted glycoprotein STC2 (stanniocalcin 2) and the proteoglycan VCAN (versican) as potential 5q oncogenes in ccRCCs. In functional assays, STC2 and VCAN each promoted tumorigenesis by inhibiting cell death. Using the same approach, we also investigated the two VHL-deficient subtypes of ccRCC, which express both HIF1α and HIF2α (H1H2) or only HIF2α (H2). This analysis revealed a distinct pattern of genomic aberrations in each group, with the H1H2 group displaying, on average, a more aberrant genome than the H2 group. Together our findings provide a significant advance in understanding ccRCCs by offering a molecular definition of two subtypes with distinct characteristics as well as two potential chromosome 5q oncogenes, the overexpression of which is sufficient to promote tumorigenesis by limiting cell death.

Interplay Between pVHL and mTORC1 Pathways in Clear-Cell Renal Cell Carcinoma

mTOR complex 1 (mTORC1) is implicated in cell growth control and is extensively regulated. We previously reported that in response to hypoxia, mTORC1 is inhibited by the protein regulated in development and DNA damage response 1 (REDD1). REDD1 is upregulated by hypoxia-inducible factor (HIF)-1, and forced REDD1 expression is sufficient to inhibit mTORC1. REDD1-induced mTORC1 inhibition is dependent on a protein complex formed by the tuberous sclerosis complex (TSC)1 and 2 (TSC2) proteins. In clear-cell renal cell carcinoma (ccRCC), the von Hippel-Lindau (VHL) gene is frequently inactivated leading to constitutive activation of HIF-2 and/or HIF-1, which may be expected to upregulate REDD1 and inhibit mTORC1. However, mTORC1 is frequently activated in ccRCC, and mTORC1 inhibitors are effective against this tumor type; a paradox herein examined. REDD1 was upregulated in VHL-deficient ccRCC by in silico microarray analyses, as well as by quantitative real-time PCR, Western blot, and immunohistochemistry. Vhl disruption in a mouse model was sufficient to induce Redd1. Using ccRCC-derived cell lines, we show that REDD1 upregulation in tumors is VHL dependent and that both HIF-1 and HIF-2 are, in a cell-type-dependent manner, recruited to, and essential for, REDD1 induction. Interestingly, whereas mTORC1 is responsive to REDD1 in some tumors, strategies have evolved in others, such as mutations disrupting TSC1, to subvert mTORC1 inhibition by REDD1. Sequencing analyses of 77 ccRCCs for mutations in TSC1, TSC2, and REDD1, using PTEN as a reference, implicate the TSC1 gene, and possibly REDD1, as tumor suppressors in sporadic ccRCC. Understanding how ccRCCs become refractory to REDD1-induced mTORC1 inhibition should shed light into the development of ccRCC and may aid in patient selection for molecular-targeted therapies.

VHL Gene Mutations and Their Effects on Hypoxia Inducible Factor HIFα: Identification of Potential Driver and Passenger Mutations

Mutations of the von Hippel-Lindau (VHL) gene are frequent in clear cell renal cell carcinomas (ccRCC). Nonsense and frameshift mutations abrogate the function of the VHL protein (pVHL), whereas missense mutations can have different effects. To identify those missense mutations with functional consequences, we sequenced VHL in 256 sporadic ccRCC and identified 187 different VHL mutations of which 65 were missense mutations. Location and destabilizing effects of VHL missense mutations were determined in silico. The majority of the thermodynamically destabilizing missense mutations were located in exon 1 in the core of pVHL, whereas protein surface mutations in exon 3 affected the interaction domains of elongin B and C. Their impact on pVHL's functionality was further investigated in vitro by stably reintroducing VHL missense mutations into a VHL null cell line and by monitoring the green fluorescent protein (GFP) signals after the transfection of a hypoxia inducible factor (HIF)α-GFP expression vector. pVHL's functionality ranged from no effect to complete HIF stabilization. Interestingly, Asn78Ser, Asp121Tyr, and Val130Phe selectively influenced HIF1α and HIF2α degradation. In summary, we obtained three different groups of missense mutations: one with severe destabilization of pVHL; a second without destabilizing effects on pVHL but relevance for the interaction with HIFα, elongin B, and elongin C; and a third with pVHL functions comparable with wild type. We therefore conclude that the specific impact of missense mutations may help to distinguish between driver and passenger mutations and may explain responses of ccRCC patients to HIF-targeted therapies.

D9S168 microsatellite alteration predicts a poor prognosis in patients with clear cell renal cell carcinoma and correlates with the down‐regulation of protein tyrosine phosphatase receptor delta

The role of microsatellite alterations in surgically excised tumors in predicting the prognosis of patients with clear cell renal cell carcinoma (ccRCC) remains largely unknown. Specimens from 93 patients with sporadic ccRCC were used to screen microsatellite alterations using 12 markers on chromosomes 3p, 9p, and 14q according to disease stage. Survival was evaluated by using the Kaplan-Meier method and Cox regression analysis. The expression of targeted genes was examined using quantitative reverse transcriptase-polymerase chain reaction and immunohistochemistry, respectively. Of the markers that were screened, D9S168 (9p23-22) had the highest frequency (36.9%) of microsatellite alteration in the specimens. D9S168 alterations were frequent in high-stage tumors. Seventy-eight patients who had ccRCC without distant metastasis at the time of surgery were followed for a median of 31.7 months. Overall survival and disease-free survival were poorer for patients with D9S168 alteration than for those without alteration (P < .001 for each; log-rank test). Cox regression analysis indicated that D9S168 alteration was associated independently with cancer-related death (P = .010). The D9S168 alteration, which was located at the 5'-untranslated region of a gene that encodes protein tyrosine phosphatase (PTP) receptor delta (PTPRD), was associated significantly with low expression of PTPRD at the messenger RNA level (P = .001). Immunohistochemistry revealed that the expression of PTPRD was strong in normal kidney proximal tubular epithelial cells, from which ccRCC originated, and was greatly down-regulated in ccRCC (P < .001; Wilcoxon rank-sum test). D9S168 microsatellite alteration in tumors predicted a poor prognosis for patients with ccRCC after curative nephrectomy. The authors concluded that PTPRD is a putative tumor suppressor in ccRCC and has therapeutic potential.

Regulatory Effects of microRNA-92 (miR-92) on VHL Gene Expression and the Hypoxic Activation of miR-210 in Clear Cell Renal Cell Carcinoma

In order to understand the role of miRNAs in renal tumorigenesis, we undertook a stepwise approach that included a comprehensive differential miRNA expression analysis for the most common histological subtypes of human renal neoplasms appearing in either sporadic or hereditary forms. We also aimed to test the hypothesis that microRNAs can act as an alternative mechanism of VHL gene inactivation and therefore might be correlated with tumorigenesis in ccRCC. Finally, we wanted to explore whether the well-known hypoxic activation of ccRCC is followed by a specific pattern of miRNA expression. Tumor and normal adjacent kidney parenchyma from patients with RCC were tested for microRNA expression. Twenty cases of different histologies were used for profiling by PCR miRNA arrays. For validation, a separate cohort of samples used to test specifically miR92a expression and its involvement in VHL gene mRNA silencing. Finally, miR210 as a marker of hypoxia was evaluated. Expression values were correlated with important clinicopathologic features from the patients. We identified unique miRNA expression signatures for each histologic subtype of kidney tumors. Expression values for downregulated miRNAs ranged from 0.3-fold (in VHL-clear cell RCC) up to 0.393 fold (in papillary type II (HLRCC) tumors). For the upregulated miRNAs, fold-changes ranged from 2.1 up to 290-fold. Specific patterns together with type-specific profiles were observed. Twenty-three miRNAs were found to be differentially expressed in both sporadic and VHL-dependent ccRCC. Sporadic clear cell tumors showed a unique pattern of 14-miRNA that were absent from the VHL-dependent tumors. These also showed 15 miRNAs specific to the hereditary type. Common miRNAs to both sporadic and hereditary forms included miR-92a and miR-210. For miR-92a, and a striking inverse correlation with VHL mRNA levels was found. For the hypoxia-regulated miR-210, clear cell tumors showed significantly higher expression levels when compared to tumor of non-clear cell histology (9.90-fold vs. 1.36, p<0.001). microRNA expression seems to be involved in every step of RCC pathogenesis: both as an element for tumor development as well as a consequence of or in response to the initial malignant transformation and part of tumor progression. Our data show consistent disregulation of miRNAs in human kidney cancer, some of which are potentially involved in critical gene silencing in RCC and others that are activated as part of the pathophysiological response in these tumors.

The impact of family history on pathological and clinical outcomes in non-syndromic clear cell renal cell carcinoma

To investigate the impact of family history on pathological and clinical outcomes after surgery for clear-cell renal cell carcinoma (ccRCC) in patients with non-syndromic disease. We reviewed 2677 patients treated with radical nephrectomy or nephron-sparing surgery for non-cystic ccRCC between 1970 and 2004 to identify patients with a family history of ccRCC. Patients with von Hippel-Lindau, tuberous sclerosis, or Birt-Hogg-Dube syndrome were excluded from analysis. Demographics and clinico-pathological outcomes were compared to patients with ccRCC without a family history of kidney cancer using chi-squared and Fisher's exact tests. Postoperative cancer-specific survival was estimated using the Kaplan-Meier method. We identified 42 patients (1.6%) with a family history of ccRCC who were treated for non-cystic ccRCC, with a median follow-up of 4.7 years (range 1-34). Demographics and tumour characteristics, including tumour stage and grade, were similar between the two groups. Patients with a family history of ccRCC were more likely to have bilateral tumours (11.9 vs 2.2%, P= 0.003). Nevertheless, cancer-specific survival rates for patients with and without a family history of ccRCC were similar at 5 years (75.7 vs 71.1%) and 10 years (53.9 vs 62.2%). Patients with a family history of ccRCC have pathological and clinical outcomes similar to patients with sporadic ccRCC. The increased incidence of bilateral tumours associated with a family history of ccRCC provides further evidence to support a nephron-sparing surgical approach when feasible.

A detailed analysis of von Hippel Lindau (VHL) mutations in sporadic clear cell renal carcinoma (ccRCC), VHL syndrome, and Chuvash polycythaemia.

e15024 Background: Germline mutations in the VHL gene cause VHL syndrome. Functional loss of the VHL gene product, pVHL is also seen in the majority of cases of sporadic ccRCC and homozygosity for the germline R200W VHL mutation causes Chuvash polycythaemia, a benign congenital erythrocytosis. pVHL has many functions including the ability to target hypoxia-inducible factor (HIF) for polyubiquitylation and proteasomal degradation by forming the substrate recognition component of a ubiquitin-ligase complex. The development of ccRCC has been attributed to impaired HIF-α downregulation by pVHL mutants. However many point mutations within VHL which are associated with ccRCC are external to the HIF-α binding site. The structural and functional effects of the R200W mutation are unknown. Methods: We present a detailed biochemical analysis of pVHL mutant proteins identified in patients with sporadic ccRCC, type 2A (low risk of ccRCC) and 2B (high risk of ccRCC) VHL syndrome and Chuvash polycythaemia. Results: Type 2A mutations Y98H and Y112H are not globally destabilising, but affect the binding site for the HIF-α hydroxyproline, as revealed by localised chemical shift changes in NMR. Mutations at S65, N78, G93 and T157 have been described in both sporadic ccRCC and type 2B VHL syndrome and are not predicted to directly impact upon the HIF-α binding site. The G93V and S65L mutations cause extensive structural perturbations of pVHL which are likely to affect both HIF-α related and HIF-α unrelated functions of pVHL. In contrast N78D, T157I and R200W mutations cause minimal structural perturbations of pVHL and do not impair HIF-α binding, implying potential novel mechanisms of pathogenicity. Truncating mutations identified in patients with sporadic ccRCC result in fundamentally unstable proteins which retain the ability to bind HIF-α, supporting previous assertions suggesting a possible dominant negative role for truncated pVHL mutants. Conclusions: Increased knowledge with respect to the functional effects of mutational events within VHL will allow us to develop informative ways of categorising these events in a way which may ultimately provide novel therapeutic opportunities. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Cancer Research UK, Medical Research Council

Abstract 330: Genomic comparison of different subtypes of renal cell carcinoma using SKY, CGH

Abstract The cytogenetic biomarkers to monitor tumor progression and treatment efficacy for renal cell carcinoma (RCC) are lacking. There are few known prognostic biomarkers that limited to tumor vascularization. In almost all sporadic clear cell RCC (ccRCC), the VHL gene in either mutated or methylated form is inactivated and often involved in cellular oxygen homeostasis. Herein, we compared different subtypes of RCC: ccRCC, chromophobe RCC (CRCC), and sarcomatoid clear cell RCC (sccRCC) in culture by using SKY and CGH. In addition, cytofluorometric analysis was performed on these tumors to give an overall immunophenotyping profile of these RCCs. By comparison of genomic characteristics of these RCC subtypes, deletion of chromosome 3p has been frequently found in sporadic ccRCC; and more complex genetic patterns of the high frequencies losses of heterozygous on chromosomes 1p, 2p, 6p, 10p, 13q, 17q, and 21q have also been documented in CRCC. However, for the sarcomatoid RCC subtype, such characteristcs have rarely been studied so far. In the current study, the genetic profile of CRCC revealed by SKY was shown: 74-84&amp;lt;4n&amp;gt; XXXX, dic(1;11)(p21;q21)×2, −3, −4, −4, der(10)(q21.1- qter), −11, −11, −18, −18, der(19)t(5;19)(q31;q?)×2, −21, −21[21]; and SCCRCC cell line showed: 40-42&amp;lt;2n&amp;gt;, X,-Y; der(2)t(2,3)(q35;q21); −3; +7,+7; der(14)(14;17)(p11;q12); −14; −15; −10; −22 [22], the data were then further confirmed by CGH for gene copy number changes of the certain specific chromosomal regions. However, for the CRCC, except loss of chromosome 21, most of genetic aberrations were different from the previous reports. On the other hand, the genetic features of current sccRCC seemed not only similar to the characteristic of ccRCC (deletion 3p), but also the genetic changes sharing the those of familial RCC (t(2;3)(q35;q21)) and papillary RCC (gain of chromosome 7 and 17, and loss of Y) documented previously. Cytofluorometric analysis revealed that there were immunophenotypic differences between CRCC and sccRCC in that surface HLA class I expression was found total loss in sccRCC, whereas CRCC expressed HLA class I abundantly on the cell surface. Furthermore, bcl-2 and URO-7 were detected in the cytoplasm of CRCC, but not in sccRCC. On the other hand, CD54 was shown on cell surface; AE3, CD44v6, Hsp70, S100-β, URO-2, URO-5, URO-9 on cell cytoplasm only observed on sccRCC. In conclusion, gain of chromosome 7 and the region harbored the oncogene c-Met were found in sccRCC, which could be related with the expression of pY1349 HGFR/c-Met associated with a high pT stage and a high metastasis rate. Therefore, it is worth to further investigate the role of c-Met and its ligand HGF in sccRCC tumorigenesis, and may help to develop clinical therapeutic strategies in sccRCC. Moreover, the immunobiological evidences clarified the significant profile of these subtypes, the information is useful on predicting the patients prognosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 330.

Alterations in VHL as potential biomarkers in renal-cell carcinoma

Germ line mutations in the VHL tumor-suppressor gene cause von Hippel-Lindau (VHL) disease, a hereditary neoplastic disease associated with clear-cell renal-cell carcinomas (ccRCCs), central nervous system hemangioblastomas and pheochromocytomas. Disruption of VHL, by somatic mutation, hypermethylation of its promoter or chromosomal loss, is also seen in the majority of cases of sporadic ccRCC. The protein product of VHL, pVHL, has multiple functions, the best-documented of which relates to its ability to target hypoxia-inducible factors (HIFs) for polyubiquitination and proteasomal degradation through its role in substrate recognition as part of a ubiquitin ligase complex. Consequently, pVHL-defective ccRCCs overexpress mRNAs that are under the transcriptional control of HIF. Drugs that modulate the downstream targets of the pVHL/HIF pathway, including sunitinib, sorafenib, temsirolimus and bevacizumab, have proven benefit in treating ccRCC. In VHL disease, clear evidence supports strong genotype-phenotype correlations, but the situation in sporadic ccRCC is less clear. Data indicate that VHL alterations have a potential role as prognostic and predictive markers in ccRCC. Future clinical trials should prospectively define the VHL alteration status of study participants so that the true utility of such markers can be determined.