Role of chromosome 3p12-p21 tumour suppressor genes in clear cell renal cell carcinoma: analysis of VHL dependent and VHL independent pathways of tumorigenesis.

AIMS: Chromosome 3p deletions and loss of heterozygosity (LOH) for 3p markers are features of clear cell renal cell carcinoma but are rare in non-clear cell renal cell carcinoma. The VHL tumour suppressor gene, which maps to 3p25, is a major gatekeeper gene for clear cell renal cell carcinoma and is inactivated in most sporadic cases of this disease. However, it has been suggested that inactivation of other 3p tumour suppressor genes might be crucial for clear cell renal cell carcinoma tumorigenesis, with inactivation (VHL negative) and without inactivation (VHL positive) of the VHL tumour suppressor gene. This study set out to investigate the role of non-VHL tumour suppressor genes in VHL negative and VHL positive clear cell renal cell carcinoma. METHODS: Eighty two clear cell renal cell carcinomas of known VHL inactivation status were analysed for LOH at polymorphic loci within the candidate crucial regions for chromosome 3p tumour suppressor genes (3p25, LCTSGR1 at 3p21.3, LCTSGR2 at 3p12 and at 3p14.2). RESULTS: Chromosome 3p12-p21 LOH was frequent both in VHL negative and VHL positive clear cell renal cell carcinoma. However, although the frequency of 3p25 LOH in VHL negative clear cell renal cell carcinoma was similar to that at 3p12-p21, VHL positive tumours demonstrated significantly less LOH at 3p25 than at 3p12-p21. Although there was evidence of LOH for clear cell renal cell carcinoma tumour suppressor genes at 3p21, 3p14.2, and 3p12, both in VHL negative and VHL positive tumours, the major clear cell renal cell carcinoma LOH region mapped to 3p21.3, close to the lung cancer tumour suppressor gene region 1 (LCTSGR1). There was no association between tumour VHL status and tumour grade and stage. CONCLUSIONS: These findings further indicate that VHL inactivation is not sufficient to initiate clear cell renal cell carcinoma and that loss of a gatekeeper 3p21 tumour suppressor gene is a crucial event for renal cell carcinoma development in both VHL negative and VHL positive clear cell renal cell carcinoma.     

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.     

Understanding familial and non-familial renal cell cancer

Molecular genetic analysis of familial and non-familial cases of conventional renal cell carcinoma (RCC) revealed a critical role(s) for multiple genes on human chromosome 3. For some of these genes, e.g. VHL, such a role has been firmly established, whereas for others, definite confirmation is still pending. Additionally, a novel role for constitutional chromosome 3 translocations as risk factors for conventional RCC development is rapidly emerging. Also, several candidate loci have been mapped to other chromosomes in both familial and non-familial RCCs of distinct histologic subtypes. The MET gene on chromosome 7, for example, was found to be involved in both forms of papillary RCC. A PRCC-TFE3 fusion gene is typically encountered in t(X;1)-positive non-familial papillary RCCs and results in abrogation of the cell cycle mitotic spindle checkpoint in a dominant-negative fashion, thus leading to RCC. Together, these data turn human RCC into a model system in which different aspects of both familial and non-familial syndromes may act as novel paradigms for cancer development.     

青少年肾细胞癌的临床病理及分子遗传学研究

目的 探讨青少年肾细胞癌的临床病理特征、遗传学改变、鉴别诊断及预后.方法 对46例青少年肾细胞癌进行光镜观察及免疫组织化学染色,随访并复习相关文献.对46例肿瘤进行von Hippel-Lindau(VHL)基因区域杂合性缺失(LOH)及VHL基因突变筛查.结果 共诊断19例Xp11.2易位/TFE3基因融合相关性肾癌(Xp11 RCC)、9例透明细胞癌、17例乳头状肾细胞癌(PRCC)和1例不能分类肾细胞癌.19例Xp11 RCC均TFE3阳性,而TFEB阴性.8例肿瘤具有巢状和乳头状结构形态类似t(X;17)ASPL-TFE3型肾癌,6例肿瘤组织学类似t(X;1)PRCC-TFE3型肾癌,4例肿瘤形态像透明细胞癌,1例肿瘤组织学形态文献中未被检索到,表现为细胞核呈毛玻璃样,核仁不明显,可见核沟,肿瘤间质见大量黏液.LOH及VHL突变检测结果显示,仅1例透明细胞癌和1例2型PRCC存在LOH,并且该2型PRCC的VHL基因的一个剪切位点存在胚系突变,553+5 G→C.其余45例均未检测出VHL突变.统计学分析表明TFE3阳性肾细胞癌比TFE3阴性肾细胞癌更倾向于高病理分期(pT3/pT4),并且预后较差(P=0.035).结论 青少年肾细胞癌表现出不同的组织学形态以及分子遗传学背景.其中Xp11 RCC为最常见的肾癌亚型.TFE3阳性肾细胞癌的预后要差于TFE3阴性肾细胞癌.     

Comprehensive mutational analysis of the VHL gene in sporadic renal cell carcinoma: relationship to clinicopathological parameters

To delineate more precisely the somatic von Hippel-Lindau disease (VHL) gene alteration as well as to elucidate its etiologic role in renal tumorigenesis, we examined a total of 240 sporadic renal cell carcinomas (RCCs) for somatic VHL gene alterations by DNA-SSCP followed by sequencing, methylation-specific PCR assay, microsatellite LOH study, and Southern blot analysis. Intragenic mutation of the VHL gene was found exclusively in clear-cell or variant-type RCCs at a frequency of 51% (104/202). Hypermethylation of the VHL promoter region was detected in an additional 11 clear-cell RCCs. Microsatellite analysis demonstrated that LOH of the VHL locus was found in 140/155 (90%) informative clear-cell RCCs. The VHL gene therefore seems to be inactivated in a two-hit manner by intragenic mutation or hypermethylation plus allelic loss in clear-cell RCC. Genomic rearrangement of the VHL gene detected by Southern analysis was not found (0/216 cases); this is in contrast to germ lines in which Southern aberrations consisted of 7-19% of the mutations. Clinicopathologic data demonstrated that VHL mutation/LOH did not vary according to tumor progression in clear-cell RCC, including tumor diameter, stage, grading, distant metastasis, and lymph node metastasis. Interestingly, VHL mutation was significantly less frequent in RCCs occurring in younger (< or = 55 years) than that in older (> or = 56 years) patients. These data suggested that the inactivation of the VHL tumor-suppressor gene is a specific genetic change in clear-cell RCC, and that it may occur at an early or first step in the clear-cell tumorigenic pathway rather than as a late event.     

Expression of HGF/SF and Met protein is associated with genetic alterations of VHL gene in primary renal cell carcinomas

We analyzed the genetic alterations of VHL, HGF/SF, and Met genes and the expression pattern of HGF/SF and Met protein in 26 renal cell carcinomas (RCCs). We found five mutations of the VHL gene and frequent LOH (50%) only in non-papillary clear cell RCC. We found six cases in which the CpG island of VHL was methylated. In addition, one missense mutation of the HGF/SF gene was detected in clear cell RCC. HGF/SF and Met protein were expressed in 84.6% and 80.7% of RCCs, respectively. All of the cases with the genetic alterations of VHL or HGF/SF demonstrated strong expression of HGF/SF and Met protein in RCC cells. Statistically, genetic alterations of VHL and HGF/SF were significantly correlated with HGF/SF and Met expression (Fisher's exact test, p=0.022 and p=0.0070). Thus, these results strongly suggest that the expression of HGF/SF and Met protein is closely associated with the genetic alterations of VHL and HGF/SF in primary RCCs.     

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。     

Characterization of a 3;6 translocation associated with renal cell carcinoma

The most frequent cause of familial clear cell renal cell carcinoma (RCC) is von Hippel-Lindau disease and the VHL tumor suppressor gene (TSG) is inactivated in most sporadic clear cell RCC. Although there is relatively little information on the mechanisms of tumorigenesis of clear cell RCC without VHL inactivation, a subset of familial cases harbors a balanced constitutional chromosome 3 translocation. To date nine different chromosome 3 translocations have been associated with familial or multicentric clear cell RCC; and in three cases chromosome 6 was also involved. To identify candidate genes for renal tumorigenesis we characterized a constitutional translocation, t(3;6)(q22;q16.1) associated with multicentric RCC without evidence of VHL target gene dysregulation. Analysis of breakpoint sequences revealed a 1.3-kb deletion on chromosome 6 within the intron of a 2 exon predicted gene (NT_007299.434). However, RT-PCR analysis failed to detect the expression of this gene in lymphoblast, fibroblast, or kidney tumor cell lines. No known genes were disrupted by the translocation breakpoints but several candidate TSGs (e.g., EPHB1, EPHA7, PPP2R3A RNF184, and STAG1) map within close proximity to the breakpoints.     

Analysis of the Birt-Hogg-Dubé (BHD) tumour suppressor gene in sporadic renal cell carcinoma and colorectal cancer

Germline mutations in the BHD gene cause the dominantly inherited cancer susceptibility disorder, Birt-Hogg-Dubé (BHD) syndrome. Individuals with BHD are reported to have an increased risk of renal cell carcinoma (RCC) and of colorectal polyps and cancer. The BHD gene maps to 17p11.2, and to investigate whether somatic inactivation of the BHD gene region is implicated in the pathogenesis of sporadic RCC and colorectal cancer (CRC), we performed mutation analysis in 30 RCC primary tumours and cell lines, and 35 CRCs and cell lines. A somatic missense mutation (Ala444Ser) with loss of the wild type allele (consistent with a two hit mechanism of tumorigenesis) was detected in a primary clear cell RCC, and a further missense mutation (Ala238Val) was identified in a clear cell RCC cell line for which matched normal DNA was not available. A somatic missense substitution (Arg392Gly) was identified in a primary CRC, and the same change was detected in three RCCs (all oncocytomas) for which matched normal DNA was not available. A germline Arg320Gln missense variant detected in a primary CRC was not detected in 40 control individuals or in a further 159 familial and sporadic CRC cases. However, AA homozygotes for an intronic single nucleotide polymorphism (c.1517+6 G-->A) were under-represented in familial cases compared with controls (p = 0.03). For some tumour suppressor genes, epigenetic silencing is a more common mechanism of inactivation than somatic mutations. However, we did not detect evidence of epigenetic silencing of BHD in 19 CRC and RCC cell lines, and BHD promoter region hypermethylation was not detected in 20 primary RCCs. These findings suggest that BHD inactivation occurs in a subset of clear cell RCC and CRC.     

VHL mutations and their correlation with tumour cell proliferation, microvessel density, and patient prognosis in clear cell renal cell carcinoma.

Mutations of the von Hippel-Lindau (VHL) gene are considered critical for the initiation of clear cell renal cell carcinoma. The VHL protein is involved in regulation of the cell cycle and neo-vascularization. In this study, the association of VHL mutations with tumour cell proliferation, angiogenesis, and clinical outcome was analysed in 113 clear cell renal cell carcinomas. The degree of angiogenesis and tumour cell proliferation was immunohistochemically determined by counting microvessels (microvessel density, anti-CD34 antibody) and cells with proliferating activity (Ki-67 labelling index, MIB-1 antibody). Forty-eight different VHL sequence alterations were found in 38 of 113 patients (34%) by direct sequencing. Nineteen VHL mutations were frameshifts and nonsense mutations, predicted to change the open reading frame of VHL. These 'loss-of-function' mutations correlated with worse prognosis in univariate analysis (p=0.02). Tumour grade, stage, microvessel density, and tumour cell proliferation were not associated with VHL alterations. These findings may indicate that 'loss-of-function' VHL mutations are involved in the progression of a clear cell renal cell carcinoma subset, whereas regulation of angiogenesis and proliferation of renal carcinoma in vivo is apparently not directly influenced by VHL alterations.     

Mutational analysis of the von hippel lindau gene in clear cell renal carcinomas from tuberous sclerosis complex patients.

Tuberous sclerosis complex (TSC) is an autosomal-dominant disorder characterized by seizures, mental retardation, autism, and tumors of multiple organs. Renal disease in TSC includes angiomyolipomas, cysts, and renal cell carcinomas. It is known that somatic mutations in the von Hippel Lindau (VHL) tumor suppressor gene occur in most clear cell renal carcinomas. To determine whether TSC-associated clear cell carcinomas also contain VHL mutations, we analyzed six tumors for loss of heterozygosity in the VHL gene region of chromosome 3p and for mutations in the VHL gene. Four of the patients were women between the ages of 34 and 68 years, and two were males under the age of 21 years. The loss of heterozygosity analysis was performed using polymorphic microsatellite markers, and the mutational analysis was performed using direct sequencing. Chromosome 3p loss of heterozygosity was not detected, and no VHL mutations were identified. These findings suggest that mutations in the TSC1 and TSC2 genes lead to clear cell renal carcinogenesis via an alternate pathway not involving VHL mutations.     

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.     

Cytogenetic and molecular analysis of early stage renal cell carcinomas in a family with a translocation (2;3)(q35;q21)

Previously, we described a family with renal cell carcinoma (RCC) and a constitutional balanced t(2;3) (q35;q21). Based on loss of heterozygosity and von Hippel-Lindau (VHL) gene mutation analyses in five tumor biopsies from three patients in this family, we proposed a multistep model for RCC development in which the familial translocation may act as a primary oncogenic event leading to (nondisjunctional) loss of the translocation-derived chromosome 3, and somatic mutation of the VHL gene as a secondary event related to tumor progression. Here, we describe the cytogenetic and molecular analysis of three novel tumors at early stages of development in two members of this family. Again, loss of derivative chromosome 3 was found in two of these tumors and a VHL mutation in one of them. In the third tumor, however, none of these abnormalities could be detected. These results underline our previous notion that loss of derivative chromosome 3 and VHL gene mutation play critical roles in familial RCC. In addition, they show that both anomalies may occur at relatively early stages of tumor development.     

Association of a novel constitutional translocation t(1q;3q) with familial renal cell carcinoma

Four cases of late onset clear cell renal cell carcinoma (RCC), a case of gastric cancer, and a case of exocrine pancreatic cancer were identified in a Japanese family. In order to elucidate the underlying mechanism for tumorigenesis in this family, extensive genetic studies were performed including routine and spectral karyotyping (SKY), fluorescence in situ hybridisation (FISH), comparative genomic hybridisation (CGH), loss of heterozygosity studies (LOH), and VHL mutation analysis. A germline translocation t(1;3)(q32-q41;q13-q21) was identified by karyotyping in five members of the family including all three RCC cases tested. The translocation was refined to t(1;3)(q32;q13.3) by FISH analysis using locus specific genomic clones, and the two breakpoints were mapped to a 5 cM region in 3q13.3 and a 3.6 cM region in 1q32. Both CGH and allelotyping using microsatellite markers showed loss of the derivative chromosome 3 carrying a 1q segment in the three familial RCCs analysed. Additional chromosomal imbalances were identified by CGH, including amplifications of chromosomes 5 and 7 and loss of 8p and 9. No germline VHL mutation was found but two different somatic mutations, a splice (IVS1-2A>C) and a frameshift (726delG), were identified in two RCCs from the same patient confirming their distinct origin.Taken together, these results firmly support a three step model for tumorigenesis in this family. A constitutional translocation t(1q;3q) increased the susceptibility to loss of the derivative chromosome 3 which is then followed by somatic mutations of the RCC related tumour suppressor gene VHL located in the remaining copy of chromosome 3.


Keywords: familial renal cell carcinoma; translocation; von Hippel-Lindau disease; loss of heterozygosity     

Somatic NF2 gene mutations in familial and non-familial vestibular schwannoma

Vestibular schwannoma occurs both as a sporadic tumour and Inthe dominantly inherited familial cancer syndrome neuroflbromatosistype 2 (NF2). The gene for NF2 has recently been Isolated onchromosome 22, and the demonstration of inactivating germllnemutations In NF2 patients and NF2 associated tumours suggeststhat it act as a tumour suppressor. We have investigated 85sporadic and 2 NF2 associated vestibular schwannomas, and onevagal schwannoma for chromosome 22 allele loss and NF2 genemutations. A further 7 vestibular schwannomas were Investigatedfor NF2 mutations only. Chromosome 22 allele loss was detectedin 34 of 87 vestibular schwannomas and In the vagal nerve schwannoma.Six exons of the NF2 gene were Investigated by SSCP analysisin all 95 tumours. Somatic NF2 gene mutations were detectedIn 13 non-familial vestibular schwannomas. and in one of theNF2 vestibular schwannomas. Seven non-famlllal tumours withan NF2 gene mutation also displayed a chromosome 22 allele loss.Thirteen of the mutations were predicted to produce truncationof the NF2 protein. These results suggest that somatic mutationsof the NF2 tumour suppressor gene are a critical step In thepathogenesis of both famlllal and non-famlllal vestibular schwannomaand that the mechanism of tumourigenesis complles with a ‘two-hit’mutation model.     

Detection of loss of heterozygosity at chromosome 3p25-26 in primary and metastatic ovarian clear-cell carcinoma: utilization of microdissection and polymerase chain reaction in archival tissues

Loss of heterozygosity (LOH) at the 3p region is found in up to 50% of epithelial ovarian neoplasms. The von Hippel-Lindau (VHL) gene at the 3p25 locus is one of the tumor-suppressor genes located at 3p. The role, if any, of the VHL gene locus is not clear in ovarian carcinogenesis. We analyzed primary and metastatic ovarian clear-cell carcinomas (OCCC) for LOH at 3p25 to determine its frequency and its diagnostic utility as an adjunctive tool in the differential diagnosis of metastatic clear-cell carcinomas. Microdissection followed by single-step DNA extraction and polymerase chain reaction (PCR) amplification, using two polymorphic markers flanking the VHL gene locus, was done on archival histology and cytology samples from 9 patients with metastatic OCCC. Of the informative cases, 43% of the metastatic and 50% of the primary OCCC showed LOH. LOH at the VHL gene locus is not uncommon in clear-cell ovarian carcinoma. LOH at 3p25 in cytologic specimens may be a valuable adjunct in the diagnosis of OCCC metastasis in cytologically equivocal cases. OCCC should enter the differential in clear-cell carcinomas of unknown primary that show LOH at 3p25. Published 2001 Wiley-Liss, Inc.     

Allelic loss on chromosomes 3p, 5q and 17p in renal cell carcinomas

Lossof-heterozygosity (LOH) has been studied on 3p (von Hippel-Lindau gene locus), 5q and 17p (p53 gene locus) by a polymerase chain reaction (PCR)-based strategy in 42 sporadic renal cell carcinomas (RCC). LOH at seven micro-satellite loci on 5q was Investigated because a tumor sup presser gene on 5q involved In the development and/or progression of RCC has not yet been identified. LOH was found In seven (17%) RCC at single or multiple locl on 5q, 38% (11/29 Informative cases) on 3p, and 6% (2/35 Informative cases) on 17p. Replication error (RER) was present in 10% (4/42) RCC at single or multiple loci. The minimum region of deletion on 5q to account for LOH was mapped to 5q31.1 (interferon regulatory factor-1; IRF-1 locus), where LOH was detected In 23% (6/26 Informative cases). LOH on 3p and 5q occurred In both stage 2 and more advanced (stage 3 and 4) tumors at similar incidences (41 and 33% on 3p; and 24 and 22% on 5q, respectively), suggesting that LOH on these chromosomes Is an early genetic event. All RCC exhibiting LOH on 3p or 5q (IRF-1 locus) were the clear cell or the mixed clear and granular cell types. These findings suggest that LOH on 3p and 5q plays an important role in the genesis of clear cell RCC. In addition, only one tumor exhibited LOH on both 3p and 5q, which suggests that LOH occurs not sequentially but independently.