Lack of mutation of the folliculin gene in sporadic chromophobe renal cell carcinoma and renal oncocytoma

Germline mutation of the folliculin gene (BHD) at chromosome 17p11.2 is associated with the development of multiplex hamartomas of the hair follicles, chromophobe renal cell carcinomas (RCC) and renal oncocytomas (RO). We have analyzed the folliculin gene with sequencing for mutations and the chromosome 17p11.2 with microsatellites for allelic changes in sporadic ROs and chromophobe RCCs. Allelic loss at chromosome 17 was seen in 8 of 8 chromophobe RCCs whereas none of the 8 RO showed alteration at this chromosomal region. Sequencing all exons from genomic DNA failed to disclose mutations of the folliculin gene in any of the tumors. We found a single nucleotide polymorphism (SNP) of G/A (nt 74) at the first exon in the untranslated region of the folliculin gene. We did not find a correlation between the SNP G/A or loss of the G allele and the expression level of either splice variants of the folliculin gene. Our data suggest the folliculin gene does not play a role in the tumorigenesis of sporadic chromophobe RCCs and renal oncocytomas.     

Sporadic human renal tumors display frequent allelic imbalances and novel mutations of the HRPT2 gene

Inactivation of the HRPT2 gene encoding parafibromin was recently linked to the familial hyperparathyroidism-jaw tumor syndrome. Patients with this syndrome carry an increased risk of parathyroid and renal tumors. To determine the relevance of HRPT2 for sporadic renal tumors, clear cell, papillary and chromophobe renal cell carcinomas as well as oncocytomas and Wilms tumors were analysed for HRPT2 gene alterations. Loss of heterozygosity (LOH) of HRPT2 was found in seven of 56 (12.5%) clear cell, three of 14 (21%) papillary, six of 10 (60%) chromophobe renal cell carcinomas, three of eight (38%) oncocytomas and four of 10 (40%) Wilms tumors. In addition, two novel HRPT2 point mutations, causing K34Q and R292K changes in parafibromin, were detected in one clear cell carcinoma and one Wilms tumor, respectively. These tumors displayed LOH of the remaining wild-type allele, but interestingly no von Hippel-Lindau (VHL) mutation. Functional analysis revealed that the K34Q mutant species of parafibromin is, unlike wild-type protein, defective in suppressing cyclin D1 expression in vivo. Taken together, these results suggest that renal cancer-associated mutations in parafibromin occur in the absence of VHL mutation, which in turn may contribute to constitutively elevated cyclin D1 expression and abnormal cell proliferation.     

S100A1 and KIT gene expressions in common subtypes of renal tumours.

AIM: The aim of this study is to evaluate the S100A1 and KIT as gene markers for the differentiation of common subtypes of renal tumours. METHODS: Fifty-five tissue samples (15 clear cell RCCs, 15 papillary RCCs, 7 chromophobe RCCs, 8 oncocytomas and 10 normal renal tissues) were studied The gene expressions of S100A1 and KIT were analysed by one-step RT-PCR by using the specific primers. RESULTS: S100A1 was expressed in 2/15 clear cell RCCs, 11/15 papillary RCCs, 7/8 oncocytomas and in 0/7 chromophobe RCCs. KIT gene was expressed in 6/7 chromophobe RCCs and 7/8 oncocytomas while 0/15 clear cell RCCs and 1/15 papillary RCCs expressed kit gene. Normal tissue expressed neither S100A1 nor KIT gene. CONCLUSION: S100A1 and KIT can be used as gene markers for the differentiation of common subtypes of renal tumours.     

Vascular cell adhesion molecule 1 predicts cancer-free survival in clear cell renal carcinoma patients.

PURPOSE: Vascular cell adhesion molecule 1 (VCAM1) is a cell surface glycoprotein implicated in various pathophysiologic conditions. We measured VCAM1 expression levels in tumor tissues and evaluated its significance and prognostic use in renal cell carcinoma (RCC). EXPERIMENTAL DESIGN: We used real-time quantitative PCR to examine the VCAM1 expression levels of a total of 485 sporadic renal tumors, including 429 clear cell, 21 papillary, 17 chromophobe, 11 oncocytomas, and 7 collecting duct carcinomas. We retrospectively examined the relationship of this expression to various clinicopathologic variables and the von Hippel-Lindau alteration status. We evaluated its significance with respect to patient survival rates using the Cox regression model combined with the split-sample method. RESULTS: Compared with normal kidney samples (n = 43), VCAM1 was significantly up-regulated in clear cell RCC and papillary RCC, whereas it was down-regulated in chromophobe RCC and oncocytoma. In clear cell RCC, VCAM1 expression levels were apparently high in patients asymptomatic at presentation and in patients with small tumor size, low-stage, low-grade, microvascular invasion-negative, and von Hippel-Lindau alteration-positive tumors. Univariate analyses showed that VCAM1 high expression is strongly associated with better outcomes in clear cell and papillary RCCs. Further, Cox multivariate analysis models combined with the split-sample method revealed that this association is significant only in cancer-free survival for patients with clear cell RCC after curative surgical resection. CONCLUSIONS: VCAM1 expression levels were found to be histologically subtype specific in renal tumors. Determination of the VCAM1 expression level as a biomarker can provide useful prognostic information for patients with clear cell RCC.     

Deletion of chromosome 3p14.2-p25 involving the VHL and FHIT genes in conventional renal cell carcinoma

Loss of heterozygosity (LOH) at chromosome 3p and inactivation of the VHL gene are associated with the development of conventional renal cell carcinomas (RCCs). Recently, it was suggested that LOH at the FHIT gene at 3p14.2 is an early event in the development of RCC and is characteristic for all types of RCC. We have analyzed 88 conventional, 30 papillary, and 22 chromophobe RCCs for LOH at the VHL and FHIT regions and at other loci on chromosome 3p. A continuous deletion of 3p14.2-p25 harboring the VHL and FHIT genes occurred in 96% of the conventional RCCs but only in 10% of the papillary RCCs and 18% of the chromophobe RCCs. Our data indicate that LOH at chromosome 3p14.2-p25 is specific for conventional RCC and that loss of one allele of both the VHL and FHIT genes occurs in early stage of tumorigenesis.     

Frequent allelic changes at chromosome 7q34 but lack of mutation of the BRAF in papillary renal cell tumors

We have analyzed the BRAF locus on chromosome 7q34 with microsatellites for allelic changes and exons 11 and 15 of the BRAF with sequencing for mutations in 50 kidney cancers including 20 papillary, 15 conventional and 15 chromophobe renal cell carcinomas (RCC). Allelic changes at the BRAF locus were seen in 16 of the 20 papillary, 3 of the 15 conventional RCCs and 2 of the 15 chromophobe RCCs. Sequencing failed to disclose mutations in exons 11 and 15 of the BRAF gene in any of the tumors. Our data indicate that BRAF mutation does not play a role in the development of renal cell tumors.     

VHL alterations in human clear cell renal cell carcinoma: association with advanced tumor stage and a novel hot spot mutation

To elucidate the role of somatic alterations for renal cancer etiology and prognosis, we analyzed 227 sporadic renal epithelial tumors for mutations and hypermethylations in the von Hippel-Lindau tumor suppressor gene VHL. Tumors were classified according to the recommendations of the Union Internationale Contre le Cancer (UICC) and the American Joint Committee on Cancer (AJCC). Somatic VHL mutations were identified by PCR, single-strand conformation polymorphism analysis, and sequencing, and hypermethylations were identified by restriction enzyme digestion and Southern blotting. Frequencies of VHL alterations were established, and an association with tumor type or tumor type and tumor stage was evaluated. VHL mutations and hypermethylations were identified in 45% of clear cell renal cell carcinomas (CCRCCs) and occasionally (3 of 28) in papillary (chromophilic) renal cell carcinomas (RCCs). Lack of VHL mutations and hypermethylations in chromophobe RCCs and oncocytomas was statistically significant (P = 0.0001 and P = 0.0004, respectively). RCCs carrying VHL alterations showed, in nine cases (12%), mutations at a hot spot involving a thymine repeat (ATT.TTT) in exon 2. Tumor staging was critical to the VHL mutation/hypermethylation detection rate in CCRCCs shown by separate evaluation of patients from medical centers in Munich, Heidelberg, and Mainz. The spectrum of pT1, pT2, and pT3 CCRCCs and the VHL mutation/hypermethylation detection rate varied among these three groups. Altogether, VHL alterations were significantly associated with pT3 CCRCCs (P = 0.009). This is the first evidence of frequent somatic VHL mutations at a particular site within exon 2 and an association of VHL mutations/hypermethylations with a standard prognostic factor.     

Differential expression of the enzyme that esterifies retinol, lecithin:retinol acyltransferase, in subtypes of human renal cancer and normal kidney.

PURPOSE: Retinoids, a group of compounds, including vitamin A (retinol), and related metabolites, have been shown to regulate the growth and differentiation of many types of cells. IFN-alpha and either 13-cis-retinoic acid or liposomal all-trans retinoic acid have been used in the treatment of patients with metastatic renal cell carcinoma. We knew that samples from renal cell carcinomas contained greatly reduced levels of retinol and retinyl esters relative to samples from normal human kidney. This prompted us to examine the levels of LRAT (lecithin:retinol acyltransferase) protein in various subtypes of human kidney cancers relative to normal human kidney by immunohistochemistry. EXPERIMENTAL DESIGN: We examined 31 partial or radical nephrectomy specimens diagnosed with kidney tumors between 1997 and 1998. Representative paraffin-embedded tissue blocks from each tumor, with each containing adjacent nonneoplastic renal parenchyma, were used for immunohistochemical analysis with affinity purified antibodies to human LRAT protein. RESULTS: LRAT protein was detected at high levels in the epithelial cells in the tubules and the lining of Bowman's capsule in the glomeruli of normal, nonneoplastic kidney sections. Among the 31 tumors, there were 13 cases of conventional (clear cell) renal cell carcinoma (RCC; including 2 multilocular cystic RCCs), 7 papillary RCC, 6 chromophobe RCC, 1 RCC, unclassified, and 4 renal oncocytoma. All tumors showed diffuse immunoreactivity for LRAT. In each case, the staining was uniform throughout the tumor, with only minimal variation in the staining intensity between different areas. All 4 renal oncocytomas, 2 of 6 chromophobe RCCs, 1 conventional (clear cell) carcinoma, 1 RCC, unclassified, and 2 conventional RCCs, which were of the multilocular cystic-type stained strongly (3+) for LRAT. In contrast, the remaining conventional RCCs and the papillary RCC samples stained much less intensely for LRAT. Of the 10 tumors that stained 3+ for LRAT in the study, 9 were either benign tumors or tumors with low malignant potential. CONCLUSIONS: These data show that LRAT expression is higher in renal tumors with an indolent biological behavior. Additional studies will ascertain if LRAT possesses any prognostic or therapeutic role in renal cancer.     

Mutations in BHD and TP53 genes, but not in HNF1beta gene, in a large series of sporadic chromophobe renal cell carcinoma

BHD, TP53, and HNF1beta on chromosome 17 were studied in 92 cases of renal cell carcinoma (46 chromophobe, 19 clear cell, 18 oncocytoma, and nine papillary). Six, thirteen, and zero cases had, respectively BHD, TP53, and HNF1beta mutations, (84% mutations involved chromophobe), suggesting a role for BHD and TP53 in chromophobe subtype.     

Distinctive expression patterns of glycoprotein non‐metastatic B and folliculin in renal tumors in patients with Birt–Hogg–Dubé syndrome

Birt–Hogg–Dubé syndrome (BHD) is an inherited disorder associated with a germline mutation of the folliculin gene (FLCN). The affected families have a high risk for developing multiple renal cell carcinomas (RCC). Diagnostic markers that distinguish between FLCN‐related RCC and sporadic RCC have not been investigated, and many patients with undiagnosed BHD fail to receive proper medical care. We investigated the histopathology of 27 RCCs obtained from 18 BHD patients who were diagnosed by genetic testing. Possible somatic mutations of RCC lesions were investigated by DNA sequencing. Western blotting and immunohistochemical staining were used to compare the expression levels of FLCN and glycoprotein non‐metastatic B (GPNMB) between FLCN‐related RCCs and sporadic renal tumors (n = 62). The expression of GPNMB was also evaluated by quantitative RT‐PCR. Histopathological analysis revealed that the most frequent histological type was chromophobe RCC (n = 12), followed by hybrid oncocytic/chromophobe tumor (n = 6). Somatic mutation analysis revealed small intragenic mutations in six cases and loss of heterozygosity in two cases. Western blot and immunostaining analyses revealed that FLCN‐related RCCs showed overexpression of GPNMB and underexpression of FLCN, whereas sporadic tumors showed inverted patterns. GPNMB mRNA in FLCN‐related RCCs was 23‐fold more abundant than in sporadic tumors. The distinctive expression patterns of GPNMB and FLCN might identify patients with RCCs who need further work‐up for BHD.     

Epigenetic inactivation of the RASSF1A 3p21.3 tumor suppressor gene in both clear cell and papillary renal cell carcinoma

Renal cell carcinoma (RCC), the most common adult kidney neoplasm, is histopathologically heterogeneous, with most sporadic RCCs ( approximately 80%) classified as clear cell (CC) tumors. Chromosome 3p allele loss is the most frequent genetic alteration in RCC but is associated specifically with sporadic and hereditary forms of clear cell RCC (CC-RCC) and is not a feature of non-CC-RCC, such as papillary (chromophilic) RCC. The VHL tumor suppressor gene (TSG) maps to chromosome 3p25, and somatic inactivation of the VHL gene occurs in up to 70% of CC-RCC tumors and cell lines. However, VHL inactivation is not sufficient for CC-RCC tumorigenesis, and inactivation of 3p12-p21 TSG(s) appears to be necessary in CC-RCC irrespective of VHL gene inactivation status. Recently, we demonstrated that the candidate 3p21 TSG, RASSF1A, is hypermethylated in most small cell lung cancers. We have now investigated the role of RASSF1A inactivation in primary RCC tumors. RASSF1A promoter methylation was detected in 23% (32 of 138) of primary CC-RCC tumors. In CC-RCC cell lines, RASSF1A methylation was associated with silencing of RASSF1A expression and restoration of expression after treatment with 5'-azacytidine. The frequency of RASSF1A methylation was similar in CC-RCC with and without VHL gene inactivation (24% versus 21%), and there was no association between epigenetic silencing of the RASSF1A and VHL TSGs, because 0 of 6 tumors with VHL hypermethylation had RASSF1A methylation, and VHL was not methylated in 26 CC-RCCs with RASSF1A methylation. Although 3p allele loss has been reported rarely in papillary RCC, we identified RASSF1A methylation in 44% (12 of 27) of papillary RCCs analyzed. Thus: (a) inactivation of RASSF1A is a frequent event in both CC-RCC and papillary RCC tumors; (b) there is no relationship between epigenetic silencing of RASSF1A and VHL inactivation status in CC-RCC. Fifty-four CC-RCCs analyzed for RASSF1A methylation were informative for 3p21 allele loss, and 20% (7 of 35) with 3p21 allele loss demonstrated RASSF1A methylation. All informative CC-RCCs with 3p21 allele loss and no RASSF1A methylation also demonstrated allele losses at other regions of 3p so that tumorigenesis in these cases may result from: (a) haploinsufficiency of RASSF1A; (b) inactivation of other 3p21 TSGs; or (c) inactivation of 3p TSGs from outside of 3p21. RASSF1A is the first TSG to be inactivated frequently in both papillary and CC-RCCs. The finding of frequent epigenetic inactivation of RASSF1A in papillary RCCs despite previous studies reporting infrequent 3p21 allele loss in this tumor type illustrates how the systematic identification of all major human cancer genes will require detailed analysis of the cancer genome and epigenome.     

Analysis of the TSC1 and TSC2 genes in sporadic renal cell carcinomas.

The genetic events involved in the aetiology of non-clear-cell renal cell carcinoma (RCC) and a proportion of clear cell RCC remain to be defined. Germline mutations of the TSC1 and TSC2 genes cause tuberous sclerosis (TSC), a multi-system hamartoma syndrome that is also associated with RCC. We assessed 17 sporadic clear cell RCCs with a previously identified VHL mutation, 15 clear-cell RCCs without an identified VHL mutation and 15 non-clear-cell RCCs for loss of heterozygosity (LOH) at chromosomes 9q34 and 16p13.3, the chromosomal locations of TSC1 and TSC2. LOH was detected in 4/9, 1/11 and 3/13 cases informative at both loci. SSCP analysis of the whole coding region of the retained allele did not reveal any cases with a detectable intragenic second somatic mutation. Furthermore, RT-PCR analysis of TSC1 and TSC2 on total RNA from 8 clear-cell RCC cell lines confirmed expression of both TSC genes. These data indicate that biallelic inactivation of TSC1 or TSC2 is not frequent in sporadic RCC and suggests that the molecular mechanisms of renal carcinogenesis in TSC are likely to be distinct.     

Mutations in the von Hippel-Lindau (VHL) gene refine differential diagnostic criteria in renal cell carcinoma

BACKGROUND AND OBJECTIVES: Renal cell carcinomas (RCC) with abundant granular cytoplasm include oncocytomas, eosinophilic variants of chromophobe RCC, papillary RCC, collecting duct carcinoma, and some conventional (clear cell) RCC. Tumors with predominantly clear cell cytoplasm include typical chromophobe RCC and conventional (clear cell) RCC. The objective of this study was to determine if mutations in the VHL gene can serve as auxiliary diagnostic criteria in refining histology based subtyping of renal epithelial neoplasia. METHODS: The study cohort of 67 cases included 24 conventional RCC, 14 chromophobe RCC, 14 papillary RCC, and 15 oncocytomas. Single strand conformational polymorphism (SSCP) was used as a screening procedure for mutations followed by automated sequencing to identify mutations. RESULTS: Thirteen of the 14 mutations identified were novel, seven of which were in the coding region. In chromophobe RCC, mutations clustered in the 5'UTR/promoter region and have not been previously reported. Exon 3 appeared to favor conventional (clear cell) RCC and correlated with a more aggressive phenotype. Mutations were absent in the papillary and oncocytoma RCC subtypes. CONCLUSIONS: Exon 3 mutations permitted a morphological distinction between conventional (clear cell) RCC and chromophobe RCC with clear cells. Mutations in the VHL gene refine histologic diagnostic criteria in RCC serving as adjuncts to the present morphology based diagnosis of RCC.     

PTEN/MMAC1/TEP1 mutations in human primary renal-cell carcinomas and renal carcinoma cell lines

Extensive allelotyping studies have implicated several tumor-suppressor loci on chromosomes 3p, 5q, 6q, 8p, 9pq, 10q, 11q, 14q, 17p, 18q and 19p in human kidney tumorigenesis. The PTEN (also called MMAC1 and TEP1) gene, a candidate tumor suppressor located at chromosome 10q23.3, is mutated in a variety of sporadic malignancies as well as in patients with Cowden disease. To investigate the potential role of the PTEN gene in renal tumorigenesis, we searched for abnormalities of the gene in 68 primary renal-cell carcinomas (RCCs) as well as in 17 renal carcinoma-derived cell lines, using DNA-SSCP, sequencing and microsatellite analysis. Five of 68 (7.5%) primary RCCs exhibited intragenic mutations (3 missense, 1 deletion and 1 splice-site), and 1 of 17 (5.9%) cell lines had an insertion mutation. Loss of heterozygosity of the PTEN gene occurred in 25% of primary RCCs, including the 3 cases with intragenic mutation and the 1 PTEN-mutated cell line. Clinical and histopathological examinations revealed that 4 of the 5 primary tumors with PTEN mutation were high-grade, advanced clear-cell RCCs with distant metastases or renal vein tumor invasions, resulting in poor prognostic courses. The other was a low-stage papillary/chromophilic RCC. Our data suggest that PTEN mutation is observed in a subset of RCCs and that, especially in clear-cell RCCs, it occurs as a late-stage event and may contribute to the invasive and/or metastatic tumor phenotype.     

Expression of KIT (CD117) in renal cell carcinoma and renal oncocytoma

OBJECTIVE: Overexpression of KIT (CD117), a tyrosine kinase receptor, has been reported in a variety of tumors, some of which are susceptible to therapy with imatinib mesylate. Our aim was to analyze KIT expression immunohistochemically in renal cell carcinomas (RCCs) and in oncocytomas. METHODS: Routinely processed, paraffin-embedded specimens from 61 RCCs and 13 renal oncocytomas were investigated immunohistochemically. Cytoplasmic and membrane-bound KIT staining of tumor cells was determined semiquantitatively. A subset of cases was additionally analyzed for point mutations of c-kit exon 17 by peptide nucleic acid-mediated nested polymerase chain reaction-clamping. RESULTS: All cases of oncocytomas and chromophobe RCCs showed membrane-bound KIT positivity, while about three-quarters of cases showed cytoplasmic reactivity. All other types of RCC were found KIT negative. Within the group of chromophobe RCCs, negative cytoplasmatic KIT reactivity was significantly correlated with advanced tumor stage (pT > or = 2; p = 0.036). Analysis of c-kit exon 17 revealed no 'gain-of-function' mutation like the codon 816 Asp-->Val mutation (D816V). Conclusions: KIT expression is a hallmark of oncocytoma and chromophobe RCC. Since all other types of RCC were found to be KIT negative, immunohistochemical KIT reactivity may be used as an additional diagnostic criterion to distinguish chromophobe RCC from other RCC types. KIT reactivity and the absence of c-kit mutation D816V in chromophobe RCC justify speculations that imatinib mesylate therapy could be effective in patients with advanced disease.     

Molecular genetics and immunohistochemistry characterization of uncommon and recently described renal cell carcinomas

Renal cell carcinoma (RCC) compromises multiple types and has been emerging dramatically over the recent several decades. Advances and consensus have been achieved targeting common RCCs, such as clear cell carcinoma, papillary RCC and chromophobe RCC. Nevertheless, little is known on the characteristics of several newly-identified RCCs, including clear cell (tubulo) papillary RCC, Xp11 translocation RCC, t(6;11) RCC, succinate dehydrogenase (SDH)-deficient RCC, acquired cystic disease-associated RCC, hereditary leiomyomatosis RCC syndrome-associated RCC, ALK translocation RCC, thyroid-like follicular RCC, tubulocystic RCC and hybrid oncocytic/chromophobe tumors (HOCT). In current review, we will collect available literature of these newly-described RCCs, analyze their clinical pathologic characteristics, discuss their morphologic and immunohistologic features, and finally summarize their molecular and genetic evidences. We expect this review would be beneficial for the understanding of RCCs, and eventually promote clinical management strategies.     

Oncofetal protein IMP3: a novel molecular marker that predicts metastasis of papillary and chromophobe renal cell carcinomas

BACKGROUND: Whether an oncofetal protein, IMP3, can serve as a prognostic biomarker to predict metastasis for patients with localized papillary and chromophobe subtypes of renal cell carcinomas (RCCs) was investigated. METHODS: The expression of IMP3 in 334 patients with primary papillary and chromophobe RCC from multiple medical centers was evaluated by immunohistochemistry. The 317 patients with localized papillary and chromophobe RCCs were further evaluated for outcome analyses. RESULTS: IMP3 was significantly increased in a subset of localized papillary and chromophobe RCCs that subsequently metastasized. Patients with localized IMP3-positive tumors (n=33; 10%) were over 10 times more likely to metastasize (risk ratio [RR], 11.38; 95% confidence interval [CI], 5.40-23.96; P<.001) and were nearly twice as likely to die (RR, 1.91; 95% CI, 1.13-3.22; P=.016) compared with patients with localized IMP3 negative tumors. The 5-year metastasis-free and overall survival rates were 64% and 58% for patients with IMP3-positive localized papillary and chromophobe RCCs compared with 98% and 85% for patients with IMP3 negative tumors, respectively. In multivariable analysis adjusting for the TNM stage and nuclear grade, patients with IMP3-positive tumors were still over 10 times more likely to progress to distant metastasis (RR, 13.45; 95% CI, 6.00-30.14; P<.001) and were still nearly twice as likely die (RR, 1.95; 95% CI, 1.15-3.31; P=.013) compared with patients with IMP3-negative tumors. CONCLUSIONS: IMP3 is an independent prognostic biomarker that can be used to identify a subgroup of patients with localized papillary and chromophobe RCC who are at high risk for developing distant metastasis.