Joerg Schubert

Analysis of genetic alterations in normal bladder urothelium

OBJECTIVES To determine whether normal mucosa had already acquired genetic changes, we analyzed the loss of heterozygosity (LOH) and chromosomal changes in normal urothelium from patients with bladder cancer and those without any history of bladder cancer. METHODS Sixteen patients with bladder cancer and 15 patients with benign prostatic hyperplasia were included in this study. Tumor tissue, as well as macroscopically normal mucosa, was examined histopathologically. We performed comparative genomic hybridization according to standard protocols to detect chromosomal alterations. Furthermore, we analyzed LOH using four markers on chromosome 9 according to standard protocols for polymerase chain reaction. RESULTS In 75% of tumor samples, LOH or shifts were detected at least with one marker on chromosome 9. Comparative genomic hybridization revealed chromosomal alterations in 12 (75%) of 16 tumors. The loss of chromosome 9 was seen frequently (56%). LOH was observed in normal mucosa in 5 of 16 patients with tumor and 1 of 15 patients with benign prostatic hyperplasia. Chromosomal alterations were also seen in the normal mucosa of 1 patient with tumor and 2 patients with benign prostatic hyperplasia (chromosomes 1, 2, 6, 14, and 17). CONCLUSIONS Our results indicated that no general genetic instability is present in the bladder urothelium. Therefore, in most patients with bladder cancer, it seems that multifocality and recurrence are not caused by genetic instability of normal urothelial cells but develop owing to cell migration or intraluminal spread.

Genetic subtyping of renal cell carcinoma by comparative genomic hybridization.

The prognosis of renal cell carcinoma (RCC) varies dependent on histologic tumor subtypes. However, differentiation between RCC types may sometimes be difficult on histologic grounds alone. Furthermore, the prognostic value of histologic parameters for the individual prognosis is limited. Additional information on the molecular level seems necessary to obtain more certainty in diagnostic and prognostic evaluation. By investigating genetic alterations in different RCC subtypes, we sought to obtain a genotype-phenotype correlation. Eighty-two clear-cell, 53 papillary, 23 chromophobe RCCs and 26 renal oncocytomas were investigated. Comparative genomic hybridization (CGH) was performed on DNA from paraffin-embedded tissue samples. DNA was isolated from tumor areas by microdissection and amplified by degenerated oligonucleotide primed polymerase chain reaction (DOP-PCR). CGH was performed according to standard protocols. We were able to detect specific alterations in each RCC subtype: clear cell RCC showed -3p, +5/5q, -8p, -9, -14, -18; papillary (chromophilic) RCC gains of chromosomes 7, 17, 16, 3, 12; chromophobe RCC loss of chromosomes 1, 2, 6, 10, 13, 17, 21; renal oncocytomas loss of chromosomes 1/1p and 14. Furthermore, for clear cell RCC, it was possible to define alterations which are associated with metastatic disease: loss of 9, 10, 14. Our results demonstrate that each RCC subtype is characterized by distinct genetic alterations. The definition of genetic alterations seems helpful for a tumor typing especially when morphology is equivocal. Therefore, genetic analyses represent a powerful diagnostic and prognostic tool for RCC.

Clonal Origin of Multifocal Renal Cell Carcinoma as Determined by Microsatellite Analysis

PURPOSE The reported incidence of satellite tumor lesions in renal cell carcinoma (7% to 25%) suggests that there is a risk of local recurrence after nephron sparing surgery. It remains largely unknown whether small satellite tumors show malignant features and whether they are metastases from the primary tumor. Therefore, we determined the clonality of multifocal tumors by molecular genetic analysis. MATERIALS AND METHODS A total of 19 multifocal clear cell renal cell carcinomas were investigated by microsatellite analysis using 6 markers for chromosome 3p, namely D3S1560, D3S1289, D3S1766, D3S1300, D3S1566 and D3S1663. Polymerase chain reaction was performed according to standard protocols, followed by gel electrophoresis and automated analysis using an automated DNA sequencer (Li-Cor, Lincoln, Nebraska). RESULTS All primary clear cell tumors were characterized by loss of heterozygosity on 3p. Multifocal tumors showed identical microsatellite alterations with at least 1 marker in 17 of the 19 cases. In 2 cases different microsatellite patterns were detected in tumors from the same kidney. CONCLUSIONS Identical loss of heterozygosity and shift patterns detected in different tumors in the same kidney strongly suggest that multifocal clear cell renal cell carcinomas have a common clonal origin in most cases. These findings indicate that satellite tumors are the result of intrarenal metastasis from the primary tumor. The clinical implications of these results must be correlated with the clinical disease course in patients with multifocal renal cell carcinoma.