Copy number profiling in von Hippel-Lindau disease renal cell carcinoma

Germline mutations in the VHL tumor suppressor gene cause von Hippel-Lindau (VHL) disease and somatic VHL mutations occur in the majority of clear cell renal cell carcinoma (cRCC). To compare copy number abnormalities (CNAs) between cRCC from VHL patients and sporadic cRCC cases without detectable somatic VHL mutations, we analyzed 34 cRCC with Affymetrix 250K arrays. To increase the power of the study, we then combined our results with those of a previously published study and compared CNAs in VHL and non-VHL related cRCC using the genomic identification of significant targets in cancer (GISTIC) program. In VHL, cRCC GISTIC analysis identified four statistically significant regions of copy number gain and four statistically significant regions of deletion that occurred in >10% of tumors analyzed. Sporadic cRCC without detectable VHL mutations had, on average, more copy number abnormalities than VHL cRCC though the most common regions of loss/gain (e.g., 3p and 14q loss and 5q gain) were present in both tumor sets. However, CNAs on chromosome arms 7p (gain) and 8p (loss) were only detected in VHL RCC. Although individual copy number abnormality peaks contained clear candidate cancer genes in some cases (e.g., the 3p loss peak in VHL cRCC contained only six genes including VHL), most peaks contained many genes. To date, only a minority of the candidate genes included in these peaks have been analyzed for mutation or epigenetic inactivation in cRCC but TNFRSF10C and DUSP4 map to the 8p region deleted in VHL cRCC and TP53 and HIF2A (EPAS1) mapped to CNA loss and gain peaks (chromosomes 17 and 2, respectively) detected in sporadic VHL wild-type cRCC.     

Metastasis of renal cell carcinoma to haemangioblastoma of the spinal cord in von Hippel-Lindau disease: case report and review of the literature

A case is presented demonstrating the unusual phenomenon of a renal cell carcinoma metastasising to a spinal haemangioblastoma in a patient with von Hippel-Lindau (VHL) disease. Recent advances in the molecular biology of VHL disease relevant to possible mechanisms of tumour-to-tumour metastasis are briefly reviewed.     

Metastatic renal cell carcinoma to hemangioblastoma in von Hippel-Lindau disease

A case of metastatic renal cell carcinoma (RCC) to a capillary hemangioblastoma (HAB) of the central nervous system in a 52-year-old woman with von Hippel-Lindau (vHL) syndrome is described. We review the literature on metastatic RCC to HAB, summarize the histologic and immunohistochemical features that can distinguish between the 2 tumors, and comment on the significance of such a finding in terms of the clinical diagnosis of vHL. We found the expression of CAM 5.2, RCC antigen, and CD10 to be strong in RCC and absent in HAB and, conversely, staining with Leu-7, neural cell adhesion molecule, and inhibin-alpha was present in HAB but weak or absent in RCC. These antibodies can be used to differentiate these entities, provided one is astute in recognizing the possibility of their coexistence.     

Germline mutation profile of the VHL gene in von Hippel-Lindau disease and in sporadic hemangioblastoma

Von Hippel-Lindau (VHL) disease is a dominantly inherited disorder predisposing those afflicted to hemangioblastomas of the central nervous system and the retina, renal cell carcinomas, pheochromocytomas, and pancreatic tumors. The disease has been associated with mutations of the VHL gene. The screening of 92 unrelated patients with VHL disease for point mutations in this gene revealed 61 DNA variants. In addition, a search for EcoR1 rearrangements revealed germline anomalies in 5 patients. The 61 variants could be subdivided in 20 mutations predicted to alter the open reading frame (8 nonsense mutations, 8 frame shift mutations, and 4 mutations in consensus splicing sites) and 43 DNA sequence variants of a priori unknown biological consequence (4 in-frame insertions or deletions, 36 missense mutations, and 3 apparently silent variations). The 3′ end of the coding sequence of the VHL gene, which encodes the Elongin binding domain was the site of 5 of 20 truncating mutations (25%) and of 18 of 41 DNA variants (44%) causing uncertain functional impairment. A similar screening in 18 patients with sporadic hemangioblastoma revealed 2 missense DNA variants. In order to corroborate this latter observation, a systematic screening for germline alteration of the VHL gene might be performed in a larger series of sporadic hemangioblastoma. If this preliminary result is confirmed, more than 10% of sporadic hemangioblastoma might be related to a mild VHL disease, thus a follow-up program similar to that recommended in cases of VHL disease should probably be discussed in the corresponding families. Hum Mutat 12:424–430, 1998. © 1998 Wiley-Liss, Inc.     

散发性肾癌中VHL基因的两个单核苷酸多态位点分析和杂合性缺失检测

目的检测79例散发性肾癌中抑癌基因VHL内部的两个单核苷酶多态(single nucleotide polymorphism，SNP)位点并分析杂合性缺失(loss of heterozygosity，LOH)发生情况，探讨VHL基因LOH与肾癌临床病理特征的关系。方法从肿瘤和正常肾组织中提取DNA，应用聚合酶链反应-限制性片段长度多态性方法检测VHL基因5′端SNP位点rs779805和3′端SNP位点rs1642742的基因型。在两个位点的杂合子中进行LOH检测，并分析VHL基因LOH与临床病理特征的关系。结果我们计算了两个位点的基因型、基因频率、杂合度、多态信息含量等遗传学参数。综合两个位点发现杂合子29例．其中12例(41．4％)存在LOH。VHL基因LOH与肾癌发生年龄、性别、临床分期、病理分级无显著相关性。结论在散发性肾癌中，VHL基因LOH是肿瘤发生的重要机理，其发生率达41．4％，VHL基因LOH与肾癌分期、分级无关。     

Metastasis of renal cell carcinoma to central nervous system hemangioblastoma in two patients with von Hippel-Lindau disease

Here we report tumor-to-tumor metastases identified in two patients with von Hippel-Lindau (VHL) disease. The first patient had bilateral renal carcinomas and multiple cerebellar hemangioblastomas, and the second patient had a renal carcinoma and multiple hemangioblastomas in the retina, cerebellum and spinal cord. A cerebellar lesion from the first patient and a spinal lesion from the second patient contained two distinct components. The inner part of these tumors consisted of a nested mass of polygonal clear cells that expressed cytokeratin and epithelial membrane antigen, while the outer part of the tumors showed proliferation of capillaries and intervening foamy stromal cells that were negative for cytokeratin and epithelial membrane antigen. The tumors were thus considered to be hemangioblastomas complicated by metastatic lesions of renal cell carcinoma of clear cell type. These cases indicate that tumor-to-tumor metastasis should be considered when hemangioblastoma contains a clear cell carcinoma component in the setting of VHL disease, and that immunohistochemical staining for cytokeratin and epithelial membrane antigen is useful for the diagnosis.     

Use of immunohistochemical methods in the differential diagnosis between primary cerebellar haemangioblastoma and metastatic renal carcinoma.

The potential role of immunohistochemistry in making the distinction between primary cerebellar haemangioblastoma and metastatic renal carcinoma was investigated by examining the reaction pattern of 10 cerebellar haemangioblastomas (seven women, three men, aged 20-40 years) and 10 primary renal carcinomas (six men, four women, aged 49-82 years) to a panel of epithelial, glial, and neural/neuroendocrine antisera. The tumour cell membranes of the renal carcinomas stained strongly with epithelial membrane antigen (EMA); membrane staining was totally absent in the haemangioblastomas. Strong neurone specific enolase (NSE) and S100 staining were also seen in haemangioblastomas but were more variable than EMA staining in renal carcinomas. It is concluded that a panel of antisera is required to distinguish between histologically similar areas in primary haemangioblastomas and metastatic renal carcinomas, and that while complementing conventional histological techniques, new problems of interpretation result which must be taken into account.     

Cytogenetics of renal cell carcinomas associated with von Hippel-Lindau disease

To establish the chromosome pattern, we have analyzed short-term cultures of 24 renal cell carcinomas (RCC) from four patients with von Hippel-Lindau disease (VHL). We evaluated the results together with those for 16 RCCs from two VHL patients karyotyped previously in our laboratory and those of 6 tumors published by others. In all 46 RCCs, the cells had lost the shortest overlapping region of the 3pl3-pter chromosome segment. The rearrangement of 3p was the only karyotype change in 20 tumors. In more than 50% of the tumors, a gain of the shortest overlapping region of the 5122-qter segment was detected. Comparative analysis showed that the chromosome aberrations in RCCs associated with VHL are similar to those found in sporadic RCCs. These results indicate that non-papillary sporadic and VHL-RCCs have common genetic mechanisms that result in the loss of the 3p13-pter region containing one or more putative suppressor genes.     

Simple renal cysts, atypical renal cysts, and renal cell carcinoma in von Hippel-Lindau disease: a lectin and immunohistochemical study in six patients

We compare the expression of four markers of renal tubular differentiation in six renal cell carcinomas, five atypical renal cysts, and five simple renal cysts from six patients with von Hippel-Lindau disease. Proximal tubular markers were expressed by five of six renal cell carcinomas, three of five atypical renal cysts, and zero of five simple renal cysts. Distal tubular markers were expressed by one of six renal cell carcinomas, five of five atypical renal cysts, and four of five simple renal cysts. One of the three atypical cysts which expressed distal tubular markers was associated with a renal cell carcinoma which also expressed distal tubular markers. Our findings suggest that simple renal cysts in von Hippel-Lindau disease arise more commonly from distal rather than proximal tubules, while atypical renal cysts show tubular origin similar to renal cell carcinomas.     

Proximal 3p deletion in renal cell carcinoma cells from a patient with von Hippel-Lindau disease

A case of proximal 3p deletion is reported in tumor cells from a patient with renal cell carcinoma and von Hippel-Lindau disease. This is the first report of this deletion with renal cell carcinoma. With the previously detected translocations at the same breakpoint, this report confirms the 3p14 breakpoint as critical in this neoplasm. This strongly suggests that renal cell carcinoma results from a change in the genetic material in the proximal short arm of chromosome #3.     

Somatic von Hippel-Lindau disease gene mutation in clear-cell renal carcinomas associated with end-stage renal disease/acquired cystic disease of the kidney

It has been documented that renal cell carcinomas (RCCs) occur frequently in patients treated with long-term dialysis, especially in cases of end-stage renal disease (ESRD)/acquired cystic disease of the kidney (ACDK). To address the molecular pathogenesis of ESRD/ACDK-associated RCCs, we examined 14 RCCs (7 clear-cell and 7 papillary carcinomas) in patients receiving dialysis for somatic mutations of the von Hippel-Lindau disease (VHL) gene as well as of the tyrosine kinase domain of the MET oncogene. Direct sequencing analyses revealed that three tumors exhibited VHL frameshifts (618delA, 386-395del10-bp, and 723-724insTC). One of the VHL mutated tumors showed additional loss of heterozygosity at the VHL gene locus. Histopathologic and clinical data demonstrated that the three tumors having VHL mutations were clear-cell RCCs occurring in ESRD with 55, 106, and 156 months of dialysis history, respectively. We did not find any tumors with mutations in the tyrosine kinase domain of the MET. These results demonstrated that the VHL tumor-suppressor gene is also involved in a subset of clear-cell RCCs occurring in ESRD/ACDK, as in the case of sporadic clear-cell RCCs. However, mutations of the MET oncogene could not be found in the seven ESRD/ACDK-associated papillary RCCs examined.     

Molecular cytogenetic characterization of early and late renal cell carcinomas in von Hippel-Lindau disease

Deletions of 3p25, gains of chromosomes 7 and 10, and isochromosome 17q are known cytogenetic aberrations in sporadic renal cell carcinoma (RCC). In addition, a majority of RCCs have loss of heterozygosity (LOH) of the Von Hippel-Lindau (VHL) gene located at chromosome band 3p25. Patients who inherit a germline mutation of the VHL gene can develop multifocal RCCs and other solid tumors, including malignancies of the pancreas, adrenal medulla, and brain. VHL tumors follow the two-hit model of tumorigenesis, as LOH of VHL, a classic tumor suppressor gene, is the critical event in the development of the neoplastic phenotype. In an attempt to define the cytogenetic aberrations from early tumors to late RCC further, we applied spectral karyotyping (SKY) to 23 renal tumors harvested from 6 unrelated VHL patients undergoing surgery. Cysts and low-grade solid lesions were near-diploid and contained 1-2 reciprocal translocations, dicentric chromosomes, and/or isochromosomes. A variety of sole numerical aberrations included gains of chromosomes 1, 2, 4, 7, 10, 13, 21, and the X chromosome, although no tumors had sole numerical losses. Three patients shared a breakpoint at 2p21-22, and three others shared a dicentric chromosome 9 or an isochromosome 9q. In contrast to the near-diploidy of the low-grade lesions, a high-grade lesion and its nodal metastasis were markedly aneuploid, revealed loss of VHL by fluorescence in situ hybridization (FISH), and contained recurrent unbalanced translocations and losses of chromosome arms 2q, 3p, 4q, 9p, 14q, and 19p as demonstrated by comparative genomic hybridization (CGH). By combining SKY, CGH, and FISH of multiple tumors from the same VHL kidney, we have begun to identify chromosomal aberrations in the earliest stages of VHL-related renal cell tumors. Our current findings illustrate the cytogenetic heterogeneity of different VHL lesions from the same kidney, which supports the multiclonal origins of hereditary RCCs. Published 2001 Wiley-Liss, Inc.     

Loss of heterozygosity on 3p in a renal cell carcinoma in von Hippel-Lindau syndrome

A renal cell carcinoma with an unbalanced t(X;3) in a patient with von Hippel-Lindau (VHL) syndrome has previously been reported. This rearrangement suggested loss of genetic material from the short arm of chromosome 3, which we are now able to confirm by restriction fragment length polymorphism analysis of tumor DNA using polymorphic probes derived from 3p. The VHL gene has recently been mapped to 3p, therefore loss of this region in this VHL-related renal cell carcinoma may have cogent significance for tumor development in this interesting cancer-predisposing syndrome.     

Mutations in the RET proto-oncogene and the von Hippel-Lindau disease tumour suppressor gene in sporadic and syndromic phaeochromocytomas.

Phaeochromocytomas may occur sporadically, or as part of the inherited cancer syndromes multiple endocrine neoplasia (MEN) type 2, von Hippel-Lindau disease (VHL), and, rarely, in type 1 neurofibromatosis. In MEN 2, germline missense mutations have been found in one of eight codons within exons 10, 11, 13, 14, and 16 of the RET proto-oncogene. In VHL, germline mutations within one of the three exons are responsible for the majority of cases. To determine if somatic mutations similar to those seen in the germline in MEN 2 or VHL disease play a role in the pathogenesis of sporadic or familial phaeochromocytomas, we analysed 48 sporadic tumours and tumours from 17 MEN 2 and five VHL patients for mutations in RET exons 9, 10, 11, 13, 14, 15, and 16, and the entire coding sequence of VHL. Five of 48 sporadic phaeochromocytomas had RET mutations within exons 10, 11, and 16. Of these, one was proven to be germline and two were proven to be somatic mutations. Four of 48 had VHL mutations; these included both the bilateral cases in the series (one was proven to be a germline mutation) and two others, of which one was proven somatic.     

Allelic deletions of the VHL gene detected in multiple microscopic clear cell renal lesions in von Hippel-Lindau disease patients.

Patients with von Hippel-Lindau (VHL) disease develop a spectrum of bilateral clear-cell renal lesions including cysts and renal cell carcinomas (RCCs). VHL gene deletions have been previously reported in VHL-associated macroscopic RCC. Although histological analysis suggests that microscopic cystic lesions in the VHL patients may represent precursors of the RCC, there is at present no direct molecular evidence of their relationship. To investigate the relationship between cystic lesions and RCC, 26 microdissected archival renal lesions from two VHL disease patients were studied for loss of heterozygosity at the VHL gene locus using polymerase chain reaction single-strand conformation polymorphism analysis. The renal lesions included 2 benign cysts, 5 atypical cysts, 5 microscopic RCCs in situ, 5 cysts lined by a single layer of cells, in which RCCs in situ were developing, and 2 microscopic and 7 macroscopic RCCs. Except for a single benign cyst, 25 of 26 renal lesions showed nonrandom allelic loss of the VHL gene. In either of the 2 patients, the same VHL allele was deleted in all of the lesions tested, indicating loss of the wild-type allele and retention of the inherited, mutated VHL allele. The results suggest that all clear-cell lesions in the VHL kidney represent neoplasms and that the loss of the VHL gene occurs early in their development. Atypical and benign cysts most likely represent the initial phenotype in malignant transformation to the RCC.     

An analysis of five clear cell papillary cystadenomas of mesosalpinx and broad ligament: four associated with von Hippel-Lindau disease and one aggressive sporadic type

Nogales F F, Goyenaga P, Preda O, Nicolae A, Vieites B, Ruiz‐Marcellan M C, Pedrosa A & Merino M J
(2012) Histopathology  60, 748–757
An analysis of five clear cell papillary cystadenomas of mesosalpinx and broad ligament: four associated with von Hippel‐Lindau disease and one aggressive sporadic type Aims: Clear cell papillary cystadenoma (CCPC) is associated with von Hippel‐Lindau disease (VHLD), but rarely involves mesosalpinx and broad ligament (M/BL). This study provides new data about its behaviour and immunophenotype. Methods and results: We performed an analysis of four benign cases of CCPC of M/BL with either characteristic clinical features or genetic markers [loss of heterozygosity (LOH)] of VHLD in patients ranging from 24 to 36 years and a sporadic case in a 52‐year‐old presenting with peritoneal metastases. All CCPCs were papillary but had solid and tubular areas. Haemorrhage, thrombosis and scarring were constant features and related to an unusual pattern of sub‐epithelial vascularity. All clear or oxyphilic cells co‐expressed cytokeratin 7 (CK7), CAM5.2 and vimentin, with strong apical CD10 and nuclear paired box gene 2 (PAX2) immunoreactivity. Three cases also showed positivity for VHL40, epithelial membrane antigen (EMA), Wilms’ tumour suppressor gene (WT‐1) and cancer antigen 125 (CA125) but only one expressed renal cell carcinoma (RCC) antigen. Vascular plexus overexpressed nuclear and cytoplasmic WT‐1. Conclusion: The VHLD‐associated cases appeared to be benign, but the sporadic case exhibited a low malignant potential. CCPCs show histological and immunophenotypical similarities with the recently reported clear cell papillary RCC, although the previously unreported apical CD10 and nuclear PAX2 expression may be related to their mesonephric origin. CCPC has a distinctive sub‐epithelial vascular pattern that is consistent with its pathogenesis.     

del(3p)(p13p21) in renal cell adenoma and del(4p)(p14) in bilateral renal cell carcinoma in two unrelated patients with von Hippel-Lindau disease

Karyotype analyses of renal cell adenoma in one patient and bilateral renal cell carcinomas (RCC) in another unrelated patient have been performed. Both patients belonged to families with von Hippel-Lindau disease (vHL). In the adenoma, we found a clonal del(3)(p13p21) and a small clone of two cells with an additional del(14)(q13). This result indicates that the same region that is often deleted in RCC may also be deleted in a renal cortical adenoma. This finding may facilitate the localization of a tentative renal cell adenoma/carcinoma tumor suppressor locus. In the tumors from the patient with bilateral carcinomas we found a clonal del(4)(p14) on one side and on the other a del(4)(p14) together with del(14)(q13). In this case, there was no detectable 3p defect in the tumors. This result raises the question whether an alternative/additional locus on chromosome 4p may be involved in the RCC/vHL syndrome. Constitutional karyotypes were in both cases normal.     

Tubular deficiency of von Hippel-Lindau attenuates renal disease progression in anti-GBM glomerulonephritis

In many kidney diseases, the original insult primarily involves the glomerulus and may then pass onto the tubulointerstitium. Several hypotheses link glomerular disease to tubular injury; perhaps the foremost hypothesis involves chronic tubular hypoxia. The reported effects of hypoxia and consecutive stabilization of hypoxia-inducible factors (HIFs), however, are controversial. Hypoxia induces interstitial fibrosis but also has beneficial effects on renal disease progression when HIF is activated pharmacologically. To analyze the impact of HIF on tubulointerstitial disease development in primary glomerular disease, transgenic von Hippel Lindau (VHL)-knockout mice were generated and null expression was induced before the onset of autoimmune IgG-mediated anti-glomerular basement membrane glomerulonephritis (GN). Tubular VHL knockout and, thus, local HIF-α stabilization increased renal production of vascular endothelial growth factor, tumor growth factor-β(1), and platelet-derived growth factor-B, resulting in augmented formation of capillaries and interstitial matrix, and conversion of fibroblasts to myofibroblasts. Within the glomerular disease, VHL knockout reduced the glomerular damage and attenuated tubulointerstitial injury. Likewise, proteinuria, plasma urea concentration, and tubulointerstitial matrix were decreased in VHL knockout with GN. These findings shown that tubular HIF-α stabilization in glomerular disease is beneficial for disease outcome. In comparison with VHL knockout alone, GN is a much stronger activator of fibrosis such that stimuli other than hypoxia may be considered important for renal disease progression.     

Clear cell carcinoid tumor of the gallbladder. A case without von Hippel-Lindau disease

A golden yellow polyp was detected in the gallbladder of a 64-year-old man who presented with epigastric pain. The lesion was composed of clear polygonal cells arranged in a trabecular and glandular pattern. The tumor invaded through the wall into the perimuscular subserosal layer. Immunohistochemical stains showed that neoplastic cells were positive for chromogranin A, synaptophysin, somatostatin, gastrin, and pancreatic polypeptide and negative for glucagon, serotonin, insulin, S100 protein, and inhibin. This tumor resembles the recently described clear cell endocrine tumors of the gallbladder and pancreas that are associated with von Hippel-Lindau disease. Our patient, however, had neither personal nor family history indicative of von Hippel-Lindau disease. Furthermore, published accounts of clear cell endocrine tumors in von Hippel-Lindau disease describe immunoreactivity for inhibin; the current case was negative for the disease. There may be a subtype of clear cell carcinoid tumor not associated with von Hippel-Lindau disease, which is characterized by its lack of immunoreactivity against inhibin.