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.     

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.     

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.     

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.     

Molecular subclassification of kidney tumors and the discovery of new diagnostic markers

We analysed the expression profiles of 70 kidney tumors of different histological subtypes to determine if these subgroups can be distinguished by their gene expression profiles, and to gain insights into the molecular mechanisms underlying each subtype. In all, 39 clear cell renal cell carcinomas (RCC), seven primary and one metastatic papillary RCC, six granular RCC from old classification, five chromophobe RCC, five sarcomatoid RCC, two oncocytomas, three transitional cell carcinomas (TCC) of the renal pelvis and five Wilms' tumors were compared with noncancerous kidney tissues using microarrays containing 19,968 cDNAs. Based on global gene clustering of 3560 selected cDNAs, we found distinct molecular signatures in clear cell, papillary, chromophobe RCC/oncocytoma, TCC and Wilms' subtypes. The close clustering in each of these subtypes points to different tumorigenic pathways as reflected by their histological characteristics. In the clear cell RCC clustering, two subgroups emerged that correlated with clinical outcomes, confirming the potential use of gene expression signatures as a predictor of survival. In the so-called granular cell RCC (terminology for a subtype that is no longer preferred), none of the six cases clusters together, supporting the current view that they do not represent a single entity. Blinded histological re-evaluation of four cases of 'granular RCC' led to their reassignment to other existing histological subtypes, each compatible with our molecular classification. Finally, we found gene sets specific to each subtype. In order to establish the use of some of these genes as novel subtype markers, we selected four genes and performed immunohistochemical analysis on 40 cases of primary kidney tumors. The results were consistent with the gene expression microarray data: glutathione S-transferase alpha was highly expressed in clear cell RCC, alpha methylacyl racemase in papillary RCC, carbonic anhydrase II in chromophobe RCC and K19 in TCC. In conclusion, we demonstrated that molecular profiles of kidney cancers closely correlated with their histological subtypes. We have also identified in these subtypes differentially expressed genes that could have important diagnostic and therapeutic implications.     

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.     

Altered expression of members of the IGF-axis in clear cell renal cell carcinoma

Renal cell carcinoma (RCC) is a heterogeneous disease and its biology is poorly understood. The commonest subtype identified is clear cell RCC. The insulin-like growth factor axis is intimately involved with many cellular roles including that of renal development. Dysregulation of this axis has frequently been demonstrated in cancer. In this study, we examine the expression of several IGF-axis components, including receptors, ligands, and binding proteins in clear cell renal cell carcinoma. A series of clear cell RCCs with matched normal kidney from the same individuals were obtained. Total RNA was extracted and expression levels of genes examined using RNase protection analysis. We confirm the dysregulation of the IGF-axis within clear cell renal cell carcinoma including the upregulation of IGFBP-3, which is further validated by immunohistochemical staining on a tissue array containing 50 RCC: positive staining in 29/30 clear cell; 1/10 papillary and 0/10 chromophobe. In addition, we demonstrate that the expression of the A isoform of the insulin receptor is significantly upregulated, while that of IGFBP-5 are significantly downregulated in this tumour 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.     

Expression of heparanase in renal cell carcinomas: implications for tumor invasion and prognosis

PURPOSE: Heparanase activity has been detected in many malignant tumors, showing a correlation with the metastatic potential. The present study was undertaken to investigate the expression of heparanase and its prognostic significance in renal cell carcinomas (RCC). EXPERIMENTAL DESIGN: Nineteen RCCs and 6 nonneoplastic renal tissues were analyzed for heparanase mRNA expression by real-time PCR. Heparanase protein expression was semiquantitatively investigated by immunohistochemistry in 70 RCCs. Involvement of heparanase in the invasiveness of RCC cell lines, 786-O and Caki-2 cells, was examined by down-regulating the gene expression with small interfering RNA (siRNA) using the Matrigel invasion assay. RESULTS: The expression level of heparanase mRNA was significantly higher in clear cell RCCs than in papillary RCCs, chromophobe RCCs, and nonneoplastic renal tissues. Heparanase was predominantly immunolocalized to cell surface and cytoplasm of clear cell RCCs and mean expression levels of heparanase were significantly higher in clear cell RCCs than in papillary and chromophobe RCCs. The protein expression levels were positively correlated with primary tumor stage, distant metastasis, and histologic grade. Targeting of heparanase mRNA expression in 786-O and Caki-2 cells with siRNA down-regulated the mRNA expression and inhibited the Matrigel invasion by these cells, whereas nonsilencing siRNA showed no effect. Multivariate Cox analysis revealed that elevated heparanase expression was a significant and an independent predictor of disease-specific survival (odds ratio, 8.814; P = 0.019). CONCLUSIONS: These data suggest that heparanase plays an important role in invasion and metastasis and silencing of the gene might be a potential therapeutic target in clear cell RCCs.     

Inactivation of BHD in sporadic renal tumors

Studies of families with Birt-Hogg-Dubé syndrome (BHD) have recently revealed protein-truncating mutations in the BHD gene, leading to tumorigenesis of the skin and of different cell types of kidney. To additionally evaluate the role of BHD in kidney tumorigenesis, we studied 39 sporadic renal tumors of different cell types: 7 renal oncocytomas, 9 chromophobe renal cell carcinomas (RCCs), 11 papillary RCCs, and 12 clear cell RCCs. We screened for BHD mutations and identified a novel somatic mutation in exon 13: c.1939_1966delinsT in a papillary RCC. We performed loss of heterozygosity (LOH) analysis on 28 matched normal/tumor sets, of which 10 of 28 (36%) demonstrated LOH: 2 of 6 (33%) chromophobe RCCs, 5 of 6 (83%) papillary RCCs, 3 of 12 (25%) clear cell RCCs, but 0 of 4 renal oncocytomas. BHD promoter methylation status was examined by a methylation-specific PCR assay of all of the tumors. Methylation was detected in 11 of 39 (28%) sporadic renal tumors: 2 of 7 (29%) renal oncocytomas, 1 of 9 (11%) chromophobe RCCs, 4 of 11 (36%) papillary RCCs, and 4 of 12 (33%) clear cell RCCs. Five tumors with methylation also exhibited LOH. Mutation and methylation were absent in 9 kidney cancer cell lines. Our results showed that somatic BHD mutations are rare in sporadic renal tumors. The alternatives, LOH and BHD promoter methylation, are the two possible inactivating mechanisms involved. In conclusion, unlike other hereditary kidney cancer-related genes (i.e., VHL and MET), which are cell type-specific, BHD is involved in the entire spectrum of histological types of renal tumors, suggesting its major role in kidney cancer tumorigenesis.     

Prognostic utility of the recently recommended histologic classification and revised TNM staging system of renal cell carcinoma: a Swiss experience with 588 tumors

BACKGROUND: A new, internationally accepted histologic classification of renal cell carcinoma (RCC) and a new edition of the TNM staging system were introduced in 1997. In the latter, there was a dramatic change in the pT classification of organ-confined renal cancer in which the break point between category pT1 and pT2 was increased from 2.5 cm to 7 cm. METHODS: To study the significance of the new pT classification and the new recommendations for histologic classification, 588 nephrectomy specimens were reevaluated to define morphologic prognostic parameters in RCC. pT classification (TNM 1997), histologic subtype, histologic tumor grade, presence of necrosis, and sarcomatoid differentiation were assessed. RESULTS: The histopathologic review according to the new classification revealed 487 conventional (clear cell) (83%), 64 papillary (11%), 31 chromophobe (5%), and 6 collecting duct (1%) RCCs. Clinical follow-up was available for 470 RCCs. The new pT classification (1997) was strongly correlated with patient survival (P < 0.0001). Histologic grade, presence of necrosis, and sarcomatoid differentiation provided independent prognostic information on the clear cell subtype of renal cancer. Sarcomatoid differentiation, but not tumor necrosis, portended a dismal prognosis for patients with papillary RCC. Chromophobe RCC was associated with a significantly better prognosis than clear cell RCC (P = 0.05). Papillary RCC with scanty cytoplasm and small cells (type 1) behaved less aggressively than papillary tumors with eosinophilic cytoplasm and large cells (type 2; P < 0.001). CONCLUSIONS: Accurate histologic classification according to the new recommendations has implications because the prognostic importance of other histologic features that are of independent significance varies with tumor subtype. The data suggest that the new pT classification allows good separation of prognostic groups of patients with renal cancer.     

Expression of cyclin D1, D3, E,and p27 in human renal cell carcinoma analysed by tissue microarray

Aberrations in the G1/S transition of the cell cycle have been observed in many malignancies and seem to be critical in the transformation process. Few studies have delineated the presence of G1/S regulatory defects and their clinical relevance in renal cell carcinoma (RCC). Therefore, we have examined the protein contents of cyclin D1, D3, E, and p27 in 218 RCCs, using tissue microarray and immunohistochemistry. The results from a subset of tumours were confirmed by Western blotting and immunohistochemical staining of regular tissue sections. Interestingly, low protein contents of cyclin D1 and p27 were associated with high nuclear grade, large tumour size, and poor prognosis for patients with conventional tumours. We further observed substantial differences in the pattern of G1/S regulatory defects between the different RCC subtypes. The majority of both conventional and papillary cases expressed p27; however, chromophobe tumours generally lacked p27 staining. In addition, conventional RCCs often expressed high cyclin D1 protein levels, while papillary RCCs exhibited high cyclin E. In summary, we have shown that G1/S regulatory defects are present in RCC and are associated with clinico-pathological parameters. The pattern of cell cycle regulatory defects also differed between RCC subtypes.     

Evolving classification of renal cell neoplasia

The development of consensus classifications for renal epithelial neoplasia in 1996 and 1997 led to the recognition of renal adenoma, renal oncocytoma and metanephric adenoma/adenofibroma as benign tumors and conventional (clear cell) renal cell carcinoma (RCC), papillary RCC, chromophobe RCC and collecting duct carcinoma as malignant morphotypes. While the overwhelming majority of renal adenomas and metanephric adenomas are benign, malignant transformation of both types have been described and genetic predictors of malignant transformation are as yet unknown. The main groups of malignant renal tumors are associated with characteristic genetic changes; conventional RCC (-3p), papillary RCC (+7, +17, -Y), chromophobe RCC (hypodiploid). Recent studies have also shown focal loss of heterozygosity of 3p segments in papillary and chromophobe RCC, indicating that 3p mutations are not confined to the conventional RCC morphotype and suggesting the presence of an important tumor suppressor gene at this site. Sarcomatoid metaplasia may occur in any morphotype and this is associated with a poor prognosis. More recently additional varieties of conventional RCC (multilocular cystic RCC), collecting duct carcinoma (medullary renal carcinoma) and papillary RCC (Types 1 and 2), each showing a characteristic morphology, have been recognized.     

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.     

Histological features of hypovascular or avascular renal cell carcinoma: the experience at four university hospitals

Background. We aimed to evaluate the pathological features of hypovascular or avascular renal cell carcinoma (RCC), by the retrospective review of the histological features that coincide with hypovascular or avascular RCC. Methods. Seven hundred and ninety-one patients who underwent both preoperative angiography and nephrectomy were examined. Of these patients, the 126 patients (15.9%) who showed hypovascular or avascular angiographic features were selected. Patients with hemorrhage or cyst(s), or with at least 50% necrosis or more in the tumor, and those with renal tumor metastatic to the kidney were excluded. The criteria proposed by the World Health Organization (1998) were adopted for the histological classification. Results. Papillary RCC was the most frequently observed hypovascular or avascular renal tumor (44 cases; 34.9%). The vascularity differed among the variants, i.e., some cases had a basophilic and solid variant with avascular features, while the remaining cases had wide stromal organization showing hypovascular features. The second most frequently observed hyporascular or avascular RCC was chromophobe cell carcinoma (35 cases; 27.8%). No difference in vascularity was detected between variants, except for 2 cases with sarcomatoid changes (avascular features). The third most frequently observed hypovascular or avascular RCC was cyst-associated RCC (29 cases; 23%). All of the 7 RCCs originating in a cyst showed avascular features, and the remaining 22 cystic RCCs showed hypovascular features. The remaining hypovascular or avascular RCCs were cases of clear cell carcinoma accompanied by sarcomatoid changes (8 cases; 6.3%), spindle cell carcinoma (5 cases; 4.0%), and collecting-duct carcinoma (5 cases; 4.0%). Conclusion. Hypovascular or avascular RCC can be categorized as non-clear cell carcinoma and some clear cell carcinoma accompanied by sarcomatoid changes. Received: December 7, 2001 / Accepted: February 18, 2002     

Evolving classification of renal cell neoplasia

The development of consensus classifications for renal epithelial neoplasia in 1996 and 1997 led to the recognition of renal adenoma, renal oncocytoma and metanephric adenoma/adenofibroma as benign tumors and conventional (clear cell) renal cell carcinoma (RCC), papillary RCC, chromophobe RCC and collecting duct carcinoma as malignant morphotypes. While the overwhelming majority of renal adenomas and metanephric adenomas are benign, malignant transformation of both types have been described and genetic predictors of malignant transformation are as yet unknown. The main groups of malignant renal tumors are associated with characteristic genetic changes; conventional RCC (-3p), papillary RCC (+7, +17, -Y), chromophobe RCC (hypodiploid). Recent studies have also shown focal loss of heterozygosity of 3p segments in papillary and chromophobe RCC, indicating that 3p mutations are not confined to the conventional RCC morphotype and suggesting the presence of an important tumor suppressor gene at this site. Sarcomatoid metaplasia may occur in any morphotype and this is associated with a poor prognosis. More recently additional varieties of conventional RCC (multilocular cystic RCC), collecting duct carcinoma (medullary renal carcinoma) and papillary RCC (Types 1 and 2), each showing a characteristic morphology, have been recognized.     

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.     

The complementary role of beta-catenin in diagnosing various subtypes of renal cell carcinomas and its up-regulation in conventional renal cell carcinomas with high nuclear grades

beta-catenin is a kind of cytoplasmic protein involved in cell adhesion and signal transduction. This study investigated its expression in various subtypes of renal cell carcinomas (RCCs) using an immunohistochemical staining method. beta-catenin expression was assessed from staining frequency and staining score. Staining score was performed by evaluating both staining percentage and intensity. All subtypes of RCCs reacted positively with beta-catenin. However, the positive frequency and staining score in papillary and chromophobe RCCs were significantly higher than those in conventional RCCs (p < 0.05). In addition, in conventional RCCs, the positive frequency and staining score of beta-catenin showed a significant difference between nuclear grades I/II and grade III (p < 0.05). Therefore, it may indicate that beta-catenin can serve as a complementary tool to distinguish conventional RCCs from chromophobe RCCs. In conventional RCCs with low nuclear grades, beta-catenin expression is generally down-regulated, while it appears to be preserved in those with high nuclear grades.