Heidi Bissig^*, Jan Richter^*, Richard Desper, Verena Meier^*, Peter Schraml^*, Alejandro A. Schäffer, Guido Sauter^*, Michael J. Mihatsch^* and Holger Moch^*

From the Institute of Pathology,^*
University of Basel, Basel, Switzerland; the National Center for Biotechnology Information,
National Institutes of Health, Bethesda, Maryland; and the Deutsches Krebsforschungszentrum,
Heidelberg, Germany
The outcome of patients with renal cell carcinoma is limited by the development of metastasis after nephrectomy. To evaluate the genetic basis underlying metastatic progression of human renal cell carcinoma in vivo, we performed a comparative genomic hybridization analysis in 32 clear-cell renal-cell carcinoma metastases. The most common losses involved chromosomes 3p (25%), 4q (28%), 6q (28%), 8p (31%), and 9p (47%). The most common gains were detected at 17q (31%) and Xq (28%). There was one high-level gene amplification at chromosome 11q22–23. The mean number of aberrations in lymph node (4.8 ± 2.8) and lung metastases (6.2 ± 4.0) was lower than in other hematogenous metastases (11.5 ± 8.7, P < 0.05), suggesting that hematogenous dissemination is linked to an acquisition of complex genomic alterations. As genetic differences between primary tumors and metastases give information on genetic changes that have contributed to the metastatic process, relative DNA sequence copy number changes in 19 matched tumor pairs were compared. Genomic changes, which frequently occurred in metastases but not in the corresponding primary tumor were losses of 8p and 9p and gains of 17q and Xq. An abnormal function of genes in these regions may contribute to the metastatic process. According to a statistical analysis of shared genetic changes in matched tumor pairs, a high probability of a common clonal progenitor was found in 11 of 19 patients (58%). Six metastases (32%) were genetically almost completely different from the primary, suggesting that detection of genomic alterations in primary tumors gives only a restricted view of the biological properties of metastatic renal cell carcinoma.

Prognosis of patients with renal cell carcinoma (RCC) is limited by the development of metastases. Five-year survival rates range from 50% to 85% in patients with organ-confined renal cancer (stage I and stage II). In contrast, less than one-third of patients with regional lymph node metastases survive 5 years. All but 5% to 10% of those patients with hematogenous metastases die within 5 years after diagnosis.¹ The most common sites of distant metastasis are lung, liver, and bones, but metastases can also develop at any other site. The metastatic behavior of RCC is often bizarre and unpredictable^2-4 .

Radiotherapy, chemotherapy, or hormonal therapy have little or no effect on metastatic RCC. Gene therapy with retroviral vector-mediated lymphokine gene transfer gave promising results in preclinical studies and is under further investigation.⁵ The metastatic cells represent the prime targets of cancer therapy. However, little is known about genetic changes with importance for the development of RCC metastases.^6-9 Chromosome 9p losses in primary RCC were associated with short metastasis-free survival.^10,11 Expression of the epidermal growth factor receptor gene¹² and the p53 gene¹³ were associated with metastatic disease.

Cancer is a genetically heterogeneous disease. Multiple clones of malignant cells are frequently detected by standard cytogenetics, by fluorescence in situ hybridization (FISH), and by flow cytometry.^14-17 Animal studies suggest that the metastatic proportions of different cell clones may be related to the metastatic process.^18,19 Therefore, chromosomal alterations responsible for metastasis may be present only in cell subpopulations of the primary tumor, which may not be detectable by molecular analyses. However, chromosomal alterations with relevance for the metastatic process should be enriched in tissue samples from metastases. Analyzing genetic changes in the metastases rather than in the more commonly targeted primary tumors could shed new light onto the molecular mechanisms of the metastatic process. Studies comparing chromosomal changes in metastases with those found in the corresponding primary tumors in the same patient would be informative in revealing differences between primary and metastatic lesions and thereby pinpointing genetic events that could have predisposed to metastatic dissemination.

Very little is known about genetic changes present in RCC metastases. Given the fact that RCC metastases may develop years or even decades after the removal of the primary tumor, genetic evolution certainly takes place within metastases. Analysis of primary tumors and their corresponding metastases allows one to assess the extent to which primary and metastatic cell clones are different from one another. A recently developed mathematical model allows testing of the statistical probability of a common clonal progenitor in primary tumors and their metastases.²⁰

To search for cytogenetic events related to metastases, 32 RCC metastases and 19 corresponding primary tumors were screened by comparative genomic hybridization (CGH). CGH allows the detection of all clonal DNA-sequence copy number aberrations (>10 MB) across the entire genome.²¹ The results implicated several genomic regions that might carry genes involved in metastasis.

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