VHL Tumor Suppressor Gene Research Articles

AB175. The first large-scale study of VHL gene mutation spectrum and genotype-phenotype correlationship of VHL disease in China

ObjectiveVHL disease is the most common hereditary renal cancer, and it is an autosomal-dominant inherited familial cancer syndrome caused by germline mutations of the VHL tumor suppressor gene. The golden standard for diagnosing VHL disease is to find the VHL gene mutation in patients’ genes. In the world, the United States had summarized 469 VHL families from the United States, Europe and Japan to summarize VHL gene mutation spectrum and the genotype-phenotype correlationship. However, no such large number of samples’ analysis had been done in China. The purpose of this study is to finish the first large-scale study of VHL gene mutation spectrum and genotype-phenotype correlationship of VHL disease in China.MethodsWe have gathered 172 Chinese VHL families including 89 families diagnosed in our hospital and 83 families from other domestic hospitals gathered by literature summary. Then we made statistical analysis of data from 172 Chinese VHL families.ResultsIn the 172 Chinese VHL families, germline mutations were identified in 165 (96%) families. Point mutations were found in 103 (62%) families, large deletions were found in 32 (19%) families, micro-deletions or micro-insertions were found in 21 (13%) families, and splice mutations were found in 9 (6%) families. We found 9 hot mutation codons that affected more than 4 families each, and they were codon65, codon76, codon78, codon80, codon86, codon88, codon161, codon162 and codon167. The onset of pheochromocytoma in VHL patients was related to mutations in VHL gene locus 482, 482, 499, 499. Mutations in VHL gene locus 257, 257, 481 was negatively related to the onset of pheochromocytoma. The occurrence of retinal angioma associated with VHL gene large deletions. Mutations in VHL gene locus 226, 233, 239, 257, 263, 481, 486, 499, 500 was related to the onset of renal cell carcinoma.ConclusionsWe have finished the first large-scale study of VHL gene mutation spectrum and genotype-phenotype correlationship of VHL disease in China. Summarizing VHL gene mutation spectrum can help to find the clinical characteristic of VHL disease and help the pathogenesis research. Genotype-phenotype correlation research can help predicting onset and evaluating prognosis of the patients.

Identification of the Lipodepsipeptide MDN-0066, a Novel Inhibitor of VHL/HIF Pathway Produced by a New Pseudomonas Species.

Throughout recent history, metabolites of microbial origin have had an extraordinary impact on the welfare of humanity. In fact, natural products have largely been –and still are– considered an exceedingly valuable platform for the discovery of new drugs against diverse pathologies. Such value is partly due to their higher complexity and chemical diversity as compared to those of synthetic and combinatorial compounds. Mutations in the Von Hippel-Lindau (vhl) gene are responsible for VHL disease, congenital polycythemia, and are found in many sporadic tumor types. The primary cause of morbidity and mortality for these patients arises from progression of Renal Cell Carcinoma (RCC) or end-stage renal disease. Inactivation of the Von Hippel-Lindau (vhl) tumor suppressor gene arises in the majority of Renal Cell Carcinoma (RCC) as well as in other types of cancer and is associated with a high degree of vascularization and poor prognosis. Loss of pVHL function thus represents a pathognomonic molecular defect for therapeutic exploitation. In this study, renal carcinoma cell lines with naturally occurring vhl mutations (RCC4 VA) and their genetically matched wild-type vhl (RCC4 VHL) counterparts were seeded onto 96-well plates and treated with a collection of 1,040 organic extracts obtained from 130 bacterial strains belonging to at least 25 genera of the phyla Actinobacteria, Firmicutes, Proteobacteria and Bacteroidetes. This strategy allowed us to identify several extracts obtained from bacterial strain F-278,770T, the type strain of the recently proposed new species Pseudomonas granadensis, showing biological activities not associated with previously known bioactive metabolites. The fractionation and structural elucidation of one of these extracts led to the discovery of a new lipodepsipeptide (MDN-0066) with specific toxicity in pVHL deficient cells that is not detectable in cells with pVHL expression rescue. This specific toxicity is associated with apoptosis induction in VHL deficient cell line as demonstrated with PARP activation and Annexin V staining. Our study demonstrated the feasibility of selectively targeting the loss of the vhl tumor suppressor gene for potential clinical benefit. Our results may have great impact on the development of new targeted therapies from natural products for the treatment of cancer and other genetic diseases.

Human renal carcinoma cells respond to Newcastle disease virus infection through activation of the p38 MAPK/NF-κB/IκBα pathway.

Newcastle disease virus (NDV) is an oncolytic virus that is known to have a higher preference to cancer cells than to normal cells. It has been proposed that this higher preference may be due to defects in the interferon (IFN) responses of cancer cells. The exact mechanism underlying this process, however, remains to be resolved. In the present study, we examined the antiviral response towards NDV infection of clear cell renal cell carcinoma (ccRCC) cells. ccRCC is associated with mutations of the von Hippel-Lindau tumor suppressor gene VHL, whose protein product is important for eliciting cellular responses to changes in oxygen levels. The most common first line treatment strategy of ccRCC includes IFN. Unfortunately, most ccRCC cases are diagnosed at a late stage and often are resistant to IFN-based therapies. Alternative treatment approaches, including virotherapy using oncolytic viruses, are currently being investigated. The present study was designed to investigate the mechanistic pathways underlying the response of ccRCC cells to oncolytic NDV infection. We found that NDV induces activation of NF-κB in ccRCC cells by inducing phosphorylation and subsequent degradation of IκBα. IκBα was found to be phosphorylated as early as 1 hour post-infection and to result in rapid NF-κB nuclear translocation and activation. Importantly, p38 MAPK phosphorylation was found to occur upstream of the NDV-induced NF-κB activation. Restoration of VHL in ccRCC cells did not result in a reduction of this phosphorylation. A similar phenomenon was also observed in several other cancer-derived cell lines. Our data provide evidence for involvement of the p38 MAPK/NF-κB/IκBα pathway in NDV infection and subsequent induction of apoptosis in ccRCC cells.

VHL gene mutation analysis of a Chinese family with non- syndromic pheochromocytomas and patients with apparently sporadic pheochromocytoma.

The Von Hippel-Lindau syndrome (VHLD), an inherited neoplastic syndrome predisposing to central nervous system hemangioblastoma (CNS), pheochromocytoma (PCC), renal cell carcinoma(RCC), retinal hemangioma (RA) and renal cysts, is caused by mutations or deletions of the VHL tumor-suppressor gene. To assess VHL genotype-phenotype correlations with function of pVHL a gene mutation analysis of members in a Chinese family with non-syndromic PCCs and individuals with apparently sporadic pheochromocytoma (ASP) was performed. DNA samples of 20 members from the Chinese family with non-syndromic PCCs and 41 patients with ASP were analyzed by polymerase chain reaction and direct sequencing, confirmed by Taqman probe. Three novel mutations (H125P, 623(?TTTGTtG) and R120T) were identified in the Chinese family and in 3 among 41 ASP patients. The mutations were all located in exon 2 of VHL gene encoding β-domain of pVHL. The tumor type in H125P carriers and R120T carriers was VHL type 2C. And 623(?TTTGTtG) carriers presented VHL type 2B or type 2C. VHL gene abnormalities were identified in the Chinese family with non-syndromic PCCs and patients with APS, resulting in dysfunction of pVHL. H125P and R120T could be associated with VHL type 2C, while 623(?TTTGTtG) might be linked with VHL type 2B or type 2C. Not only is the genetic analysis helpful for early diagnosis and treatment of patients with VHLD, it is also benefitial for research into VHLD pathogenesis.

Abstract 3438: Integrated analysis of germline and somatic variants in renal clear cell carcinoma

Abstract Each year in the United States, there are approximately 54,000 new cases of kidney and upper urinary tract cancers and 13,000 deaths. Renal clear cell carcinoma (RCC) is the most common type of kidney cancer comprising more than 75% of cases. It is known that certain genetic syndromes strongly increase risk for renal cell carcinoma including Von Hippel Lindau disease (VHL) that is caused by germline mutations in the VHL tumor suppressor gene. However, there are a large number of cases with familial RCC that are not explained by germline VHL mutations. Publically available data generated through large scale genomic sequencing projects such as The Cancer Genome Atlas (TCGA) provides new opportunities for identifying both known and novel germline cancer susceptibility variants. Moreover, the availability of matched tumor and normal genomic sequencing data from cancer patients (including RCCC) through TCGA allows for integrated analyses of germline and somatic interactions. Toward this end, we analyzed the TCGA exome sequencing data from 499 RCC cases. We employed GATK, VarScan, and Pindel tools to identify germline single nucleotide variants and indels and identified 10,492 candidate germline rare truncation variants (X truncation variants were in cancer genes). All the truncation candidates in commonly mutated cancer driver genes were manually reviewed, resulting in a total of 119 high confidence germline truncation variants in cancer genes. To date, we have identified one nonsense mutation in the VHL gene. In addition, there was one nonsense mutation and a frame-shift deletion in WRN and a nonsense and a splice-site mutation in BAP1, a recently identified RCC predisposition gene. Moreover, we also identified 6,079 candidate missense variants in known cancer driver genes, among a total of 179,066 rare missense variants. Future directions for this project will be to use the burden analysis to determine genes with significant enrichment for deleterious variants. We will also examine loss of heterozygosity patterns to evaluate germline somatic interactions. Citation Format: Jiayin Wang, Charles Lu, Mingchao Xie, Piyush Tripathi, Michael McLellan, Feng Chen, Kimberly J. Johnson, Li Ding. Integrated analysis of germline and somatic variants in renal clear cell carcinoma. [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 3438. doi:10.1158/1538-7445.AM2014-3438

Surgical Management of Sporadic Spinal Cord Hemangioblastomas

Hemangioblastomas are benign, slow growing but highly vascularized tumors of the central nervous system, with the most common location of occurrence in the posterior fossa. Hemangioblastomas usually have an associated with patients that have Von-Hippel Lindau disease, resulting a germline mutation in the VHL tumor suppressor gene. Isolated or sporadic occurrences of hemangioblastomas are much more infrequent and typically respond well after surgery.We present case of a 22 year old female with worsening shoulder pain, decreased sensation in the hands and feet, and decreasing strength and was found to have a hemangioblastoma of the cervical spine.The patient was treated with surgery and responded well to treatment. We also present a review of the literature on isolated occurrences of hemangioblastomas of the spinal cord.Isolated hemangioblastoma are a rare tumor of the central nervous system and can be managed with surgery.

Phase 3 randomized study of cabozantinib (XL184) versus everolimus in subjects with clear cell renal cell carcinoma (METEOR).

TPS4601 Background: Cabozantinib is an oral receptor tyrosine kinase inhibitor (TKI) with potent activity against MET and VEGFR2. Cabozantinib has demonstrated clinical activity in multiple solid tumor types, including heavily pretreated subjects with clear cell renal cell carcinoma (ccRCC) (Choueiri et al.; ASCO 2012, abstract 4504). In ccRCC, VHL tumor suppressor gene dysfunction results in impaired HIF degradation and consequent activation of MET and VEGF pathways. Moreover, MET has been implicated in adaptive resistance to VEGF-pathway targeted agents. Based on the clinical activity seen to date and the scientific rationale, there is strong interest in the evaluation of cabozantinib in ccRCC. Methods: This Phase 3, open-label, multicenter study evaluates the efficacy and safety of cabozantinib compared with everolimus in subjects with ccRCC (NCT01865747).The primary endpoint is progression-free survival (PFS) as evaluated by an independent radiology review committee (IRC). Secondary endpoints are over...

HIF1α deubiquitination by USP8 is essential for ciliogenesis in normoxia

Loss of primary cilia is a key feature of von Hippel-Lindau tumor suppressor (VHL)-associated pathology. Although VHL-deficiency predisposes cells to precipitous cilia disassembly in response to growth factor cues, it does not affect ciliogenesis. Here, using a siRNA-based screen to find genes that are essential for ciliogenesis only in the presence of the VHL tumor suppressor gene product pVHL, we identify ubiquitin-specific protease (USP)8. The pVHL-dependency of USP8 for ciliogenesis is directly linked to its function as a HIF1α deubiquitinating enzyme. By counteracting pVHL-mediated ubiquitination of HIF1α, USP8 maintains a basal expression of HIF1α and HIF transcriptional output in normoxia, including the repression of Rabaptin5, which is essential for endosome trafficking-mediated ciliogenesis.

SQSTM1 Is a Pathogenic Target of 5q Copy Number Gains in Kidney Cancer

Clear cell renal cell carcinoma (ccRCC) is the most common form of kidney cancer and is often linked to loss of chromosome 3p, which harbors the VHL tumor suppressor gene, loss of chromosome 14q, which includes HIF1A, and gain of chromosome 5q. The relevant target(s) on chromosome 5q is not known. Here, we show that 5q amplification leads to overexpression of the SQSTM1 oncogene in ccRCC lines and tumors. Overexpression of SQSTM1 in ccRCC lines promoted resistance to redox stress and increased soft agar growth, while downregulation of SQSTM1 decreased resistance to redox stress, impaired cellular fitness, and decreased tumor formation. Therefore, the selection pressure to amplify 5q in ccRCC is driven, at least partly, by SQSTM1.

Methylation profiling and evaluation of demethylating therapy in renal cell carcinoma.

BackgroundDespite therapeutic advances in targeted therapy, metastatic renal cell carcinoma (RCC) remains incurable for the vast majority of patients. Key molecular events in the pathogenesis of RCC include inactivation of the VHL tumour suppressor gene (TSG), inactivation of chromosome 3p TSGs implicated in chromatin modification and remodelling and de novo tumour-specific promoter methylation of renal TSGs. In the light of these observations it can be proposed that, as in some haematological malignancies, demethylating agents such as azacitidine might be beneficial for the treatment of advanced RCC.ResultsHere we report that the treatment of RCC cell lines with azacitidine suppressed cell proliferation in all 15 lines tested. A marked response to azacitidine therapy (>50% reduction in colony formation assay) was detected in the three cell lines with VHL promoter methylation but some RCC cell lines without VHL TSG methylation also demonstrated a similar response suggesting that multiple methylated TSGs might determine the response to demethylating therapies. To identify novel candidate methylated TSGs implicated in RCC we undertook a combined analysis of copy number and CpG methylation array data. Candidate novel epigenetically inactivated TSGs were further prioritised by expression analysis of RCC cell lines pre and post-azacitidine therapy and comparative expression analysis of tumour/normal pairs. Thus, with subsequent investigation two candidate genes were found to be methylated in more than 25% of our series and in the TCGA methylation dataset for 199 RCC samples: RGS7 (25.6% and 35.2% of tumours respectively) and NEFM in (25.6% and 30.2%). In addition three candidate genes were methylated in >10% of both datasets (TMEM74 (15.4% and 14.6%), GCM2 (41.0% and 14.6%) and AEBP1 (30.8% and 13.1%)). Methylation of GCM2 (P = 0.0324), NEFM (P = 0.0024) and RGS7 (P = 0.0067) was associated with prognosis.ConclusionsThese findings provide preclinical evidence that treatment with demethylating agents such as azacitidine might be useful for the treatment of advanced RCC and further insights into the role of epigenetic changes in the pathogenesis of RCC.

VHL Gene Alterations in Italian Patients with Isolated Renal Cell Carcinomas

Clear cell renal cell carcinoma (ccRCC) is the most common malignant neoplasm of the kidney and belongs to the few human tumors known to develop from mutations of the VHL tumor suppressor gene. VHL germline mutations are associated with hereditary ccRCCs in VHL disease. However, somatic VHL gene defects may also occur in sporadic ccRCCs. In this study, we analyzed the frequency and the spectrum of VHL gene alterations in 35 Italian patients with sporadic renal cell carcinoma (RCC). Tumor-specific intragenic VHL pathogenic mutations were detected in 38% (11/29) of the ccRCC patients and 33% (2/6) of the patients with other types of RCC. One novel 18-bp in-tandem duplication and 4 previously unreported nucleotide changes in the VHL gene were described. Microsatellite analysis showed loss of heterozygosity for at least 1 informative marker in 43% (9/21) of the ccRCCs and 50% (3/6) of the non-ccRCCs; 5 of the 13 tumors (38%) harboring VHL gene alterations also had loss of heterozygosity for at least 1 microsatellite marker. Our results confirm that somatic inactivation of the VHL gene may play a pivotal role in the tumorigenesis of sporadic ccRCCs in Italian patients and suggests that mutation analysis of the VHL gene may be helpful for discriminating sporadic, VHL-gene-related ccRCCs from those related to VHL disease.

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.

655 ISOLATED PHAECHROMOCYTOMA

Introduction: Phaechromocytoma is a neuroendocrine tumor associated with a variety of genetic disorders wich include(VHL),multiple endocrine neoplasia type2,Neurofibromatosis type 1 and hereditary pargangliomas.These hereditary syndrome are respectively caused by respectively mutation in VHL tumor suppressor gene,RET protooncogene,NF1 gene and gene coding succinate deshydrogenase SDHB,SDHC and SDHD.It is currently estimated that 20% of phaechromacytoma are associated with germline mutation.VHL predispose affected individual to develop retinal and central nervous hemangioblastoma, renal celle carcinoma and phaechromocytoma. We report a novel VHL germ-line mutation in a family with occurrence of isolated phaechromocytoma. Case: The 38 years old, caucasian male was admitted in hospital for major weakness, sweating ; body loss,elevated pressure, moderate renal failure ;Hypokalaemia. Plasma renine aldosterone activity is high, plasma fractionnal free metanephrine is very high Proteinuria :3 gr/24 h, microscopic haematuria. urinary normetadrenaline was very high 15916ng/24 h (normal < 200). Radiologic test: Brain, spinal, abdominal and thoraco abdominal CT scan, showed bilateral surrenale mass, left measured 4.5*3.5*4 cm, right 4.5*4*4.4 cm .MIBG scintigraphy : cervical para ganglioma. Patient have had bilateral adrenalectomy. Genetic analysis: VHL germline mutation was identified in the patient and family members.The c.640T > A mutation detected in our reported family has not been previously described. Discussion: This patient had presented phaechromocytoma in association and without other manifestation of VHL disease. The association of this mutation (c.640 > A) with phaechromocytoma only, variant of VHL disease, might be considered. To confirm this suggestion it seems reasonnable to subject these patient to lifelong follow up.

Abstract 2170: The SWI/SNF nucleosome-remodeling gene PBRM1 - Another tumor suppressor gene in renal cell carcinomas

Abstract The polybromodomain containing gene PBRM1 was recently discovered to harbor 41% truncating somatic mutations in clear cell renal cell carinomas (ccRCC). Next to the VHL tumor suppressor gene this gene is thought to be the second major cancer gene in ccRCCs. PBRM1 (also called BAF180) belongs to the SWI/SNF-B or PBAF complex and provides functional specificity to this class of chromatin remodelers by binding to acetylated histones via its six bromodomains, two BAH domains, and one HMG domain (the latter binding to nucleosomal DNA). PBRM1 is located on chromosome 3p21 in close vicinity to VHL and SETD2 genes arguing for a putative gatekeeper function in cases where VHL loss alone is insufficient for ccRCCs tumorigenesis. PBRM1 codes for at least three major transcripts of about 7500nt in length whose expression was measured by Q-PCR in 49 cases of paired normal and tumor tissue samples of RCC patients who underwent radical tumor nephrectomy at our clinic between 06/2009 and 10/2010. 20/49 cases showed a marked upregulation of PBRM1 expression whereas in 29/49 cases PBRM1 was clearly downregulated. To assess the contribution of somatic mutations for PBRM1 downregulation we resequenced 29 PBRM1 exons of 48 tumor tissues. Specific truncating mutations were detected in 6 (15.4%) of 39 ccRCCs. These mutations include 5 deletions in exons 1, 10, 16, 23, and 24, as well as one insertion in exon 16. All these heterozygous mutations lead to a decrease of PBRM1 mRNA expression, that was verified by Q-PCR measurements. In addition, 6 coding SNPs (cSNPs) were revealed as well as 5 SNPs in intronic regions, one of the latter is in close vicinity to the splice acceptor site of exon 9. Concurrent expression of three major transcripts (var. 1,2, and 4) is seen in all 46 matched normal and tumor samples analyzed so far. In 22 (47.8%) samples we detected a dominance of var.1 (NM_018165) in normal tissue when compared to the corresponding tumor sample, that preferentially expresses var.4 (NM_181042). So far our data set of 15.4% truncating mutations in 39 cases of ccRCC could not match the exceptional high number of 41% reported in the initial report by the Sanger Institute. Truncating mutations of PBRM1 have been described in breast cancers but were absent in lung carcinomas, despite the fact that LOH at 3p21 is frequently seen in these cancers. Understanding the contribution of PBRM1 expression changes, be it on the genomic level (mutation) and/or mRNA (differential splicing), to clinical disease progression and outcome in kidney cancer needs further investigation. 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 2170. doi:1538-7445.AM2012-2170

Extraadrenal Pheochromocytoma and Vagal Paraganglioma

Pheochromocytomas and catecholamine-secreting paragangliomas, also referred to as extraadrenal pheochromocytomas, are rare neuroendocrine tumors that arise from chromaffin cells of the adrenal medulla and sympathetic ganglia. These tumors are uncommon causes of hypertension, accounting for <0.2% of cases (1, 2). While pheochromocytomas are inherently catecholamine-secreting tumors, paragangliomas are less likely to be functional, especially when located in the head and neck. Paragangliomas are rare and widespread tumors, commonly found in the head and neck region. The most common location for head and neck tumors is at the carotid bifurcation, while abdominal tumors are most commonly found in the periaortic-pericaval region (3). While most cases are sporadic, about one fourth of patients with pheochromocytomas are found to have genetic mutations associated with familial disorders, including multiple endocrine neoplasia type 2 syndrome associated with the RET protooncogene, von Hippel-Lindau syndrome associated with the VHL tumor suppressor gene, and mutations in succinate dehydrogenase subunits B and D. These patients typically present at a younger age and are more likely to have bilateral adrenal pheochromocytomas or paragangliomas (4). Paragangliomas are also associated with inherited syndromes caused by genetic mutations in succinate dehydrogenase subunits. Succinate dehydrogenase inactivation has been implicated with the activation of hypoxia-inducible genes involved with tumorigenesis and is an active area of cancer research (5). Rates of malignancy for pheochromocytomas have been reported as 5% to 26%, while reports of malignant paragangliomas are more rare (6, 7). Malignant tumors lack histological and molecular markers that can reliably distinguish them from benign tumors (8). In addition, malignant tumors classified as benign may present at a later time with widely metastatic disease and associated decreased overall survival (9). We present the case of a 51-year-old man with an extraadrenal pheochromocytoma and vagal paraganglioma diagnosed within several months of each other. The patient was successfully treated with surgical excision. While pheochromocytomas and paragangliomas are well documented, to our knowledge there are few if any case reports of an extraadrenal pheochromocytoma and a vagal paraganglioma in the same patient. Therefore, this case report should inform the practitioner of the possibility of multiple primary tumors when treating patients with either condition.

Renal cell Cancer, Environmental Arsenic Exposure and Carcinogenic Mutations

Malignant tumors of the kidney accounts for approximately 2-3% of the all new primary cancers cases diagnosed in the US, with an estimated 60.000 cases occurring annually. Current epidemiological studies provide convincing evidence linking environmental arsenic exposure to an elevated risk of renal cell carcinoma (RCC). High levels of arsenic leaching from natural underground sources have contaminated newly drilled wells, leading to cancers of the skin, lung, liver, kidney, and bladder. This link has been the basis for regulatory actions. In 2002, the US Environmental Protection Agency (EPA) lowered the maximum contaminant level for the amount of arsenic allowed in drinking water from 50 ppb to 10 ppb (10 µ/L). In addition, recent molecular biology research on familial kidney cancer syndromes indicate that inactivation mutation in VHL tumor suppressor gene contributes to clear cell RCC development. Future studies using animal models are needed to explain genetic susceptibility in VHL mutations and carcinogenic effect of arsenic exposure.

Bilateral Pheochromocytoma in von Hippel-Lindau Syndrome Simultaneously Removed by Lateral Retroperitoneal Endoscopic Approach

ABSTRACT Von Hippel-Lindau (VHL) syndrome is an autosomal dominant familial neoplastic syndrome caused by mutation in VHL tumor suppressor gene localized on chromosome 3p25. The disease is characterized by abnormal vascular proliferation and increased risk of developing renal cell carcinoma, pheochromocytoma, hemangioblastoma of the central nervous system, tumors of the endolymphatic bag, cysts of the kidney, liver and pancreas, epididymal cystadenomas, neuroendocrine tumors of the pancreas, angiomas in the retina. We report a case of a bilateral pheochromocytoma, simultaneously removed by unilateral total and contralateral subtotal retroperitoneal endoscopic adrenalectomy. How to cite this article Todorov G, Grozdev K, Lukanova T, Mioljevikj-Miserliovska B, Miserliovski R. Bilateral Pheochromocytoma in von Hippel-Lindau Syndrome Simultaneously Removed by Lateral Retroperitoneal Endoscopic Approach. World J Endoc Surg 2012;4(3):102-104.

Abstract B126: Optimization of culture conditions for the establishment of de novo renal cell carcinoma cell lines.

Abstract Renal cell carcinoma, a common malignancy of the genitourinary tract, accounts for approximately 85% of all renal malignancies. Clear cell RCC (ccRCC) is the most common subtype and has a high level of cellular heterogeneity, with a characteristic mutation in the von Hippel-Lindau (Vhl) tumor suppressor gene in a majority of ccRCC cases. While extirpative surgery is most often curative in tumors restricted to the kidney parenchyma, locally advanced and metastatic ccRCC is resistant to conventional chemotherapeutics and radiation therapy. Several aspects of ccRCC biology, including mechanisms of progression, tumor heterogeneity, and identification of biomarkers and candidate therapeutic targets are being examined. However, these studies typically use cell lines cultured in serum-containing media and passaged for extended periods of time. These lines acquire mutations in vitro, irreversibly corrupting their genotype and phenotype compared to the corresponding patient's tumor. Recent studies in the glioma field have shown that cell lines established in serum-free media maintained their ex vivo phenotype and genotype, and enabled more relevant functional screens like RNA knockdown and chemical cytotoxicity screens. More recently, similar approaches have been used to derive more biologically relevant serum-free cancer cell lines in other cancers, but not in ccRCC. We established a number of lines directly from patient tumors using traditional fetal bovine serum containing culture conditions in ambient (20%) oxygen versus serum-free media containing defined growth factor under 2% oxygen to minimize reactive-oxygen-species-induced mutations. In order to validate both their origin as cancer cells, and maintenance of their genotype and phenotype during cell culture, we used a panel of ten matched cell line pairs (serum and serum-free) and assessed a) cell line and tumor fidelity by STR profiling, b) genomic conservation by copy number variation, and c) Vhl mutation status. All of the cell lines were found to match the primary patient tissue by STR profiling. Two of the ten lines grown in serum recapitulated the patient tumor Vhl mutation, but none of the lines grown in serum-free media had any mutations characteristic of the tumour, and were thus derived from contaminating normal kidney epithelial cells. These findings suggest that normal cells out-grow ccRCC cells under both culture conditions, but serum-containing media may be more permissive for ccRCC growth. We did not observe any accumulation of additional genetic changes in either culture condition. Our Vhl-mutant cell line fails to survive in the serum-free culture conditions used here, thus providing us with a tool for the optimization of serum-free growth conditions and identification of factors necessary for in vitro growth and survival of ccRCC cells. These conditions will then be used to derive additional lines that may represent more accurate in vitro models of ccRCC. Furthermore, molecular characterization of the matched wild type and mutant lines will allow us to identify markers to more easily distinguish normal cells from cancer cells in vitro. We believe it is important to identify a defined serum-free culture condition that supports ccRCC cell growth, as this will lead to identification of molecules that are essential for ccRCC cell growth and survival, and thus act as potential therapeutic targets. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B126.

Genetic and functional studies implicate HIF1α as a 14q kidney cancer suppressor gene.

Kidney cancers often delete chromosome 3p, spanning the VHL tumor suppressor gene, and chromosome 14q, which presumably harbors ≥ 1 tumor suppressor genes. pVHL inhibits the hypoxia-inducible transcription factor (HIF), and HIF2α is a kidney cancer oncoprotein. In this article, we identify focal, homozygous deletions of the HIF1α locus on 14q in clear cell renal carcinoma cell lines. Wild-type HIF1α suppresses renal carcinoma growth, but the products of these altered loci do not. Conversely, downregulation of HIF1α in HIF1α-proficient lines promotes tumor growth. HIF1α activity is diminished in 14q-deleted kidney cancers, and all somatic HIF1α mutations identified in kidney cancers tested to date are loss of function. Therefore, HIF1α has the credentials of a kidney cancer suppressor gene. Deletion of 14q is a frequent event in clear cell renal carcinoma and portends a poor prognosis. In this study, we provide genetic and functional evidence that HIF1α is a target of 14q loss in kidney cancer.

Identification of mutations of the SWI/SNF complex gene PBRM1 by exome sequencing in renal carcinoma.

4571 Background: The genetics of renal cancer is dominated by inactivation of the VHL tumour suppressor gene in clear cell carcinoma (ccRCC), the commonest histological subtype. Methods: A recent large-scale screen of ~3500 genes by PCR-based exon re-sequencing identified several new cancer genes in ccRCC including UTX (KDM6A), JARID1C (KDM5C)and SETD2. These genes encode enzymes that demethylate (UTX, JARID1C) or methylate (SETD2) key lysine residues of histone H3. Results: Modification of the methylation state of these lysine residues of histone H3 regulates chromatin structure and is implicated in transcriptional control. However, together these mutations are present in fewer than 15% of ccRCC, suggesting the existence of additional, currently unidentified cancer genes. Here, we have sequenced the protein coding exome in a series of primary ccRCC and report the identification of the SWI/SNF chromatin remodeling complex gene PBRM1 as a second major ccRCC cancer gene, with truncating mutations in 41% (92/227) of cases. Conclusions: These data further elucidate the somatic genetic architecture of ccRCC and emphasize the marked contribution of aberrant chromatin biology.