Recalling Cohnheim's Theory: Papillary Renal Cell Tumor as a Model of Tumorigenesis from Impaired Embryonal Differentiation to Malignant Tumors in Adults.

We have suggested that papillary renal cell tumor (PRCT) of the kidney arises from nephrogenic rest-like lesions. To approve our hypothesis, we worked up 14 kidneys bearing papillary and 14 ones with conventional renal cell carcinoma (CRCC) histologically and found 42 papillary lesions in average per kidney bearing PRCT. PRCTs are characterized by loss of the Y chromosome and trisomy of chromosomes 7 and 17. The MET and HNF1B are localized to chromosome 7q31 and 17q21 and are frequently amplified in PRCT. We have analyzed the expression of the mutant MET in hereditary PRCTs and precursor lesions and found duplication and expression of the mutated allele. Because both genes are involved in early stage of nephron development, we have analyzed the expression of MET and HNF1B by immunohistochemistry in fetal kidneys, precursor lesions and PRCTs. We detected strong expression of MET and HNF1B in distal compartment of S-shaped body of fetal kidneys and in nephrogenic rest-like precursor lesions. Our finding suggests an association between expression of MET and HNF1B in precursor lesions and development of PRCT. We propose a model involving chromosomal clonal evolution and corresponding gene expression for development of PRCTs from embryonic rests due to impaired differentiation. Our model suggests that PRCT have a natural history distinct from that of most common CRCC.

High risk of development of renal cell tumor in end-stage kidney disease: the role of microenvironment.

End-stage renal disease (ESRD) and acquired cystic renal disease (ACRD) are associated with high risk of development of renal cell tumors (RCT) displaying unusual phenotype and genotype. The underlying molecular mechanism is not yet known. To explore the molecular microenvironment, we have established the expression profile of ESRD/ACRD kidneys. RNA extracted from normal and ESRD/ACRD kidneys and distinct types of RCT was subjected to Affymetrix HG U133 micro array analysis. A gene expression signature indicated cancer-related biological processes in the remodeling of ESRD/ACRD kidneys. Quantitative RT-PCR studies confirmed a specific gene signature including a functional group of inflammatory cytokines and also cytokeratins associated with stem cell characteristics of epithelial cells. Several of the signature genes including the SCEL were expressed in ESRD/ACRD-associated papillary RCT as well. Immunohistological analysis confirmed the expression of CXCL8 and its receptor CXCR2 as well as the expression of SCEL in hyperplastic tubular, cystic, and papillary structures and RCTs in ESRD/ACRD kidney. Our data indicates that ESRD/ACRD is a novel disease and the inflammatory microenvironment altered plasticity, and stem cell characteristics of epithelial cells may be associated with the high risk of tumor development.

Sciellin is a marker for papillary renal cell tumours.

There are no adequate immunohistochemical markers for papillary renal cell tumours. The aim of this study was to establish a gene expression profile of papillary renal cell tumours using an expression microarray approach. Through hierarchical clustering and significant analysis of microarrays, we have selected the best 13 genes and analysed their expression by real-time polymerase chain reaction (RT-PCR). Of these genes, we selected SCEL as potential marker of interest. Immunohistochemical staining of tissue microarrays containing all major types of kidney cancers revealed positive staining for sciellin in 87 of 114 papillary renal cell tumours and in 13 of 19 precursor lesions. No other renal tumour types were positive for sciellin. Our study indicates that although not all tumours express sciellin, its expression may help to confirm the diagnosis papillary renal cell tumour.

P53 mutation and LOH at chromosome 9 in urothelial carcinoma.

BACKGROUND: Mutation of the p53 gene has been implicated in the development of carcinoma in situ (CIS) to invasive solid urothelial carcinomas (UC) whereas loss of heterozygosity (LOH) at chromosome 9 has been suggested to plag part in the development of papillary UCs. PATIENTS AND METHODS: The p53 mutation and LOH at chromosomes 17p13.1 and 9 were analysed in 120 UCs. Tumor and matched normal DNA were used for microsatellite allelotyping of chromosome 17p and the entire chromosome 9. RESULTS: LOH at 17p13.1 was found in each grade and stage of the UCs, but mutation of the p53 occurred only in the highly malignant G3 tumors including papillary pT1G3 UCs. LOH were found at one or more of the seven tumor suppressor gene loci along chromosome 9 in all but two of the UCs with p53 mutation. CONCLUSION: Mutation of the p53 gene is not a pathway correlated genetic change, but is associated with the increased cell proliferation of G3 UCs.

Molecular analysis of germline t(3;6) and t(3;12) associated with conventional renal cell carcinomas indicates their rate-limiting role and supports the three-hit model of carcinogenesis.

We describe the molecular analysis of chromosomal rearrangements in familial t(3;6)(p12.3;q24.3) and t(3;12)(q13.13;q24.23) associated with the development of conventional renal cell carcinomas (RCC). We mapped the breakpoints by high-density oligo array comparative genomic hybridization of tumor cells in t(3;6) at chromosome 3p12.3 between PDZRN3 and CNTN3; the chromosomal rearrangement at 6q24.3 was mapped within the seventh intron of the STXBP5 gene. In the second case, the break at 3q13.13 was mapped downstream of PVRL3 and the breakpoint at 12q24.23 between HSPB8 and CCDC60, one allele of the latter being deleted. Reverse transcriptase polymerase chain reaction analysis of the PDZRN3, CNTN3, STXBP5, PVRL3, HSPB8, and CCDC60 genes revealed slight variation in the copy number of transcripts, but without correlation to the chromosomal rearrangements in translocation-associated and sporadic conventional RCCs. Loss of heterozygosity at chromosome 3p and mutation of VHL occurred at the same frequency in both familial and sporadic cases. Based on our model of nonhomologous chromatid exchange and the data on molecular studies, we suggest that the germline translocation serves as a rate-limiting step toward tumor development by generating a high number of cells with loss of the derivative chromosome carrying the 3p segment.

Oligoarray comparative genomic hybridization of renal cell tumors that developed in patients with acquired cystic renal disease.

Renal cell carcinoma occurs at higher frequency in acquired cystic renal disease than in the general population. We have analyzed 4 tumors obtained from the kidneys of 2 patients with acquired cystic renal disease, including 2 conventional renal cell carcinomas and 2 acquired cystic renal disease-associated tumors, for genetic alterations. DNA changes were established by applying the 44K Agilent Oligonucleotide Array-Based CGH (Agilent Technologies, Waldbronn, Germany), and mutation of VHL gene was detected by direct sequencing of the tumor genome. DNA losses and mutation of the VHL gene, which are characteristic for conventional renal cell carcinomas, were seen in 2 of the tumors. The acquired cystic renal disease-associated eosinophilic-vacuolated cell tumor showed gain of chromosomes 3 and 16. No DNA alterations occurred in the papillary clear cell tumor. We suggest that not only the morphology but also the genetics of renal cell tumors associated with acquired cystic renal disease may differ from those occurring in the general population.

Phosphatemia is related to chromosomal aberrations of parathyroid glands in patients with hyperparathyroidism.

BACKGROUND AND AIMS: It has been well documented that gene and DNA alterations occur frequently in benign primary parathyroid adenomas as well as in parathyroid glands with secondary hyperplasia. However, it has not been shown whether a correlation exists between somatic DNA aberrations and clinical data. METHODS: We analyzed the frequency of chromosomal aberrations in adenomas obtained from 25 patients with primary hyperparathyroidism (pHPT) and 60 parathyroid nodules from 20 uremic patients with secondary hyperparathyroidism (sHPT). The relation of chromosomal aberrations to parathyroid hormone, as well as calcium and phosphate serum concentrations, was assessed. Allelic changes were evaluated by microsatellite allelotyping using 105 polymorphic markers. RESULTS: Somatic chromosomal aberrations were found in 23 out of 25 adenomas, in hyperplastic lesions from 16 out of 20 patients. In pHPT as well as in sHPT a positive correlation was found between the number of chromosomal alterations and serum phosphate concentration (tau=0.270, p=0.05; and tau=0.362, p=0.03, respectively). Only in pHPT was a negative correlation of borderline significance between serum parathormone (PTH) and number of aberrated chromosomes noticed (tau=-0.258, p=0.07). There was no correlation between the number of DNA changes and serum concentration of calcium or tumor volume. CONCLUSION: Hyperphosphatemia may increase the risk of specific and random chromosomal aberrations due to increasing proliferation rate of parathyroid cells in patients with sHPT.

Cloning and characterisation of the RBCC728/TRIM36 zinc-binding protein from the tumor suppressor gene region at chromosome 5q22.3.

DNA alterations at chromosome 5q22 occur frequently in different types of tumors including urological cancers. Previously, we narrowed a common target region between loci D5S659 and D5S2055 at chromosome 5q22.3 by microsatellite allelotyping. After constructing a BAC contig and shot-gun sequencing we identified a putative exon by the NIX software package. By PCR cloning using the putative exon (5qex5) specific primers and primers directed to the vector (lambdaZAPII) sequence of human brain and kidney cDNA libraries, we obtained a full-length cDNA of 4074 bp of the new gene RBCC728/TRIM36 (GenBank accession no.) with an ORF coding for a protein of 728 amino acids. The TRIM36, which is a new member of the tripartite motif (TRIM) gene family, shows a RING finger C3HC4 structure, two B-box, a coiled-coil, a fibronectin type III and a C-terminal domain of unknown function (SPRY). The TRIM36 has a weak homology to MID1. Immunohistochemistry of recombinant and native TRIM36 displays a cytoplasmic, slightly filamentous staining pattern in COS-7 cells. The TRIM36 is expressed in adult testis, brain, prostate, kidney, heart and lung. A variable level of TRIM36 expression was detected by Q-RT-PCR in conventional RCC, while the gene was consequently upregulated in PCs. We did not find mutation in the open reading frame of the TRIM36 in cancer cells. The overexpression of the TRIM36 in the vast majority of prostate cancer suggest that this gene might be involved in the prostate tumorigenesis.

Mutations of mtDNA in renal cell tumours arising in end-stage renal disease.

Toxic effects in the uraemic state or during maintenance dialysis have been suggested to be responsible for DNA damage and tumour development in end-stage renal disease (ESRD). This study therefore analysed the mitochondrial DNA alterations in six kidneys with ESRD and in nine renal cell tumours arising in these kidneys. Sequencing the entire 16 569 bp mitochondrial genome disclosed 94 sequence variations in normal and corresponding tumour tissues. Thirty-eight polymorphisms occurred in the D-loop region, 40 in the polypeptide coding regions, 12 in the rRNAs, and four in the tRNAs. Nine somatic nucleotide changes were found in seven of the nine tumours analysed; four of them were G to A transitions. Two of the G to A changes occurred in the D-loop region, one in the MTTA gene, and one in the MTND2 gene. An A to G substitution was seen in the control region at the mtTF1 binding site. A T to C transition also occurred also in the D-loop region. A T insertion was seen in MTRNR2 (16S rRNA). One C insertion in MTND4 and one A deletion in the polyA tract of the MTND5 gene resulted in frameshift mutations in two tumours. This study reveals a high mutational rate of the mitochondrial DNA in tumours, which may correspond to the increased level of reactive oxidative species in renal parenchymal cells in ESRD.

Somatic mitochondrial DNA mutations in human chromophobe renal cell carcinomas.

We sequenced the entire mitochondrial genome in 8 chromophobe renal cell carcinomas (RCCs) and corresponding normal kidneys. Our study disclosed 68 known and 45 new sequence variations occurring 132 and 45 times, respectively. We found 6 somatic nucleotide changes in 5 out of the 8 chromophobe RCCs. One A --> T substitution occurred in the D-loop region and an insertion of a 9-bp sequence in the noncoding region of the MTNC7. One G --> A substitution and one C --> T substitution were seen in the MTRNR1 and MTRNR2 genes, respectively. One C deletion in MTND5 and one T insertion in the MTND3 gene resulted in frameshift mutations in two tumors. All somatic alterations, with the exception of the 9-bp insertion, were heteroplasmic changes. Although somatic mtDNA mutations are found in chromophobe RCCs, their role in the maintenance of tumor cell phenotype or in tumorigenesis remains to be elucidated.