The expression of G250/mn/CA9 antigen by flow cytometry: its possible implication for detection of micrometastatic renal cancer cells.

Monoclonal antibody (mAb) G250 is a well characterized and specific mAb to renal cell carcinoma (RCC). The gene G250 was recently cloned and was proved to be homologous to MN/CA9. The G250/MN/CA9 antigen was recently explored as a potential marker for RCC. Flow cytometry (FCM) allows quantitative analysis of cells. The present study describes a flow cytometric method to detect this antigen in human cell lines and in malignant and normal renal tissues. Twelve human carcinoma cell lines (HeLa, Colo205, HT29, BxPC3, OVCAR3, SKOV3, ACHN, A704, CAKI-2, SKRC-59, SKRC-10, and SKRC-52), 10 specimens of normal peripheral blood mononuclear cells, and 38 malignant and 36 adjacent normal renal tissues were studied. The malignant and normal renal tissues were disaggregated mechanically into a single-cell suspension, stained by mAb G250, and analyzed by FCM. All 22 of the clear cell carcinomas, 6 of 8 mixed cell carcinomas, and 3 of 6 granular cell carcinomas were positive for G250/MN/CA9 antigen. SKRC-52 and SKRC-10 were strongly positive for G250/ MN/CA9. The G250/MN/CA9 antigen could also be detected in HeLa, SKOV3, HT29, and A704 cells. One chromophobic, one chromophilic cell carcinoma, the normal renal tissues, and normal peripheral blood mononuclear cells were considered as negative. Our results further confirmed that the G250/MN/CA9 antigen was an ideal marker for RCC, especially for clear cell carcinomas, and that this antigen was present in several types of malignant cells. FCM may serve as a fast tool of immunocytochemical detection of renal cancer cells. Flow cytometric detection of renal cancer cells by using mAb G250 should be further explored.     

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.     

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.     

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.     

Overexpression of KIT in chromophobe renal cell carcinoma

We analysed gene-expression profiles in 15 surgical specimens of conventional, papillary, and chromophobe renal cell carcinomas (RCCs) using high-density oligonucleotide arrays. From about 12,000 genes targeted by the array, 67 were upregulated specifically in each histological type of RCC. The oncogene KIT was one of the genes whose expression was upregulated specifically in chromophobe RCCs. Immunohistochemical analysis demonstrated the KIT gene product on the cell membrane of chromophobe RCC in all cases, although it was not detected in conventional RCCs or non-neoplastic kidneys except for weak staining in the cytoplasm of renal tubules. These results suggest that each histological subtype of RCC has a unique gene-expression profile, and in particular indicates for the first time that KIT could be a useful marker for chromophobe RCC. As overexpression of KIT might be involved in tumor growth, KIT could be a new therapeutic target in this special type of RCC.     

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.     

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.     

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.     

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.     

Can renal oncocytomas be distinguished from renal cell carcinoma on fine-needle aspiration specimens? A study of conventional smears in conjunction with ancillary studies.

BACKGROUND: The reliability of using fine-needle aspiration (FNA) to distinguish renal oncocytoma (RO), a benign tumor, from renal cell carcinoma (RCC), which has eosinophilic granular cytoplasm, has been questionable. However, it is clinically significant, because radical nephrectomy may be avoided in patients with RO. The authors retrospectively studied the cytologic features and ancillary study findings of RO compared with findings in RCCs with eosinophilic granular cytoplasm to evaluate the reliability of FNA-based diagnosis of RO. METHODS: The authors reviewed 19 tumors, including 11 ROs, three chromophobe RCCs (CRCCs), three granular variant RCCs (GRCCs), and two eosinophilic variant papillary RCCs (EPRCCs). Smears and cell blocks were prepared using either computed tomography-guided or ultrasound-guided FNA material. Surgical specimens were available for all tumors. Cytokeratin, vimentin, and Hale colloidal iron (HCI) stains were performed on all 19 tumors. Electron microscopy (EM) was available for six tumors. RESULTS: Although most tumors demonstrated their classic cytologic features, the specific diagnosis using conventional smears or even core biopsies was difficult in some tumors, especially ROs, due to the overlapping cytomorphology among these tumors. Cytologic material was obtained from 10 of 11 RO specimens. Of 10 ROs, 8 original FNA-based diagnoses were oncocytic neoplasm. Immunoperoxidase studies revealed that all tumors of each type were positive for cytokeratin, whereas only GRCCs and EPRCCs were positive for vimentin. The two vimentin negative neoplasms, RO and CRCC, could be distinguished by HCI stain, which showed diffuse or focal cytoplasmic positivity in CRCCs and apical/perinuclear staining (73%) or negative staining (27%) in ROs. Ultrastructurally, cytoplasm densely packed with mitochondria was characteristic for oncocytoma. CONCLUSIONS: This study demonstrated that ROs can be distinguished reliably from RCCs on the basis of cytologic morphology combined with ancillary studies, including immunostaining with cytokeratin and vimentin antibodies and HCI stain. EM provides additional information to confirm the diagnoses.     

Histologic coagulative tumor necrosis as a prognostic indicator of renal cell carcinoma aggressiveness

BACKGROUND: Prognostic markers for renal cell carcinoma (RCC), such as patient symptoms, tumor stage, tumor size, and tumor grade, are useful for determining appropriate follow-up and selecting patients for adjuvant therapy. Histologic coagulative tumor necrosis, also reported to be a prognostic marker for RCC, has not previously been extensively described or investigated. Hence, the objective of the current study was to characterize tumor necrosis as a prognostic feature of RCC. METHODS: The authors of the current study identified 3009 patients treated surgically for RCC between 1970 and 2002 from the Mayo Clinic Nephrectomy Registry (Rochester, MN). Associations of tumor necrosis with clinical, laboratory, and pathologic features were examined with chi-square, Fisher exact test, and Wilcoxon rank-sum tests. Cancer-specific survival was estimated with the Kaplan-Meier method, and associations with outcome were assessed with Cox proportional hazard models. RESULTS: Tumor necrosis was present in 690 of 2445 (28%) clear cell, 196 of 421 (47%) papillary, and 28 of 143 (20%) chromophobe RCCs. The risk ratio for death from RCC in patients with necrotic compared with non-necrotic tumors was 5.27 (95% confidence interval [CI]: 4.56-6.09; P < 0.001) for clear cell, 4.20 (CI: 1.65-10.68; P < 0.001) for chromophobe, and 1.49 (CI: 0.81-2.74; P = 0.199) for papillary RCC. The survival difference for clear cell RCC persisted even after multivariate adjustment for tumor stage, size, and grade (risk ratio 1.90; P < 0.001). CONCLUSIONS: Histologic coagulative tumor necrosis is an independent predictor of outcome for clear cell and chromophobe RCC, and it should be routinely reported and used in clinical assessment.     

Prognostic relevance of global histone H3 lysine 4 (H3K4) methylation in renal cell carcinoma

Epigenetic alterations play an important role in carcinogenesis. Recent studies suggested that global histone modifications are predictors of cancer recurrence in various tumor entities. Our study was performed to evaluate histone H3 lysine 4 mono‐methyl (H3K4me1), ‐di‐methyl (H3K4me2) and ‐trimethyl (H3K4me3) patterns in renal cell carcinoma (RCC) using a tissue microarray with 193 RCC (including 142 clear cell, 31 papillary, 10 chromophobe and 10 sarcomatoid RCC) and 10 oncocytoma specimens: H3K4me3 staining was more intense in papillary RCC, whereas H3K4me1 and H3K4me2 were similar in the diverse RCC subtypes. H3K4me2 and H3K4me3 levels were increased in oncocytoma. H3K4me1‐3 levels were inversely correlated with Fuhrman grading, pT stage, lymph node involvement and distant metastasis. Progression‐free survival and cancer‐specific survival were shorter in patients with low levels of H3K4me1‐3 in the univariate analysis, but we did not observe a significant correlation of a single modification in a multivariate model, which also included the established prognostic parameters TNM‐stage and Fuhrman grade. In comparison, the H3K4me score, which combined staining levels of the H3K4 modifications, was an independent predictor of RCC progression‐free survival. Our study on H3K4 methylation supports the concept of global histone modifications as potential cancer prognosis markers.     

Hypomethylation of the MN/CA9 promoter and upregulated MN/CA9 expression in human renal cell carcinoma

MN/CA9 is a cancer-related gene, frequently activated in human renal cell carcinomas (RCCs). To reveal the activation mechanism, we investigated the relationship between methylation status of the MN/CA9 promoter region and gene expression using 13 human RCCs, and examined the effect of in vitro CpG methylation on the MN/CA9 promoter activity using a human RCC cell line (SK-RC-44), expressing MN/CA9. MN/CA9 expression was evaluated by RT-PCR and observed in 10 of 13 RCCs (77%). A total of 9 out of 10 MN/CA9 -positive RCCs (90%) contained clear cell components. Methylation status of 6 CpGs in the MN/CA9 promoter region was decided by using the bisulfite genomic sequencing protocol. Out of 13 RCCs 9 (69%) showed partial hypomethylation of the CpG at -74 bp, while the other 4 RCCs and 3 normal kidney tissue samples showed complete methylation. Hypomethylation of the CpG at -74 bp was strongly correlated with MN/CA9 expression. Luciferase assay revealed that the MN/CA9 promoter activity was strongly suppressed by methylation of the CpG at -74 bp. These findings suggest that hypomethylation of the CpG at -74 bp in the MN/CA9 promoter region might play an important role in this gene activation of human RCC.     

肾嗜酸细胞瘤螺旋CT特征及鉴别要点分析

目的：观察肾嗜酸细胞瘤的平扫及动态增强扫描特征,并与肾嫌色细胞癌进行比较。方法选择20例肾嗜酸细胞瘤患者为研究对象,另选择30例肾嫌色细胞癌患者为对照组,观察肾嗜酸细胞瘤CT表现,比较肾嗜酸细胞瘤与肾嫌色细胞癌病变形态学特征,病灶强化百分比及肿瘤-肾皮质强化指数。结果肾嗜酸细胞瘤密度于皮髓质期、实质期及排泄期均低于肾脏皮质,差异均有统计学意义（P﹤0.05）;肾嗜酸细胞瘤密度于皮髓质期、肾实质期及排泄期高于肾脏髓质,差异具有统计学意义（P﹤0.05）;肾嗜酸细胞瘤较肾嫌色细胞癌体积小,密度均匀的比例高,存在星芒状瘢痕的比例高,差异均具有统计学意义（P﹤0.05）。动态增强扫描,肾嗜酸细胞瘤与肾嫌色细胞癌强化程度间比较显示,肾嗜酸细胞瘤在皮髓质期、实质期的病灶强化百分比和肿瘤-肾皮质强化指数,均高于肾嫌色细胞癌,差异均具有统计学意义（P﹤0.05）。结论肾嗜酸细胞瘤的CT表现具有一定的特征,形态学特征及动态增强表现有助于鉴别诊断。