Loss of heterozygosity and copy number abnormality in clear cell renal cell carcinoma discovered by high-density affymetrix 10K single nucleotide polymorphism mapping array

Abstract

Genetic aberrations are crucial in renal tumor progression. In this study, we describe loss of heterozygosity (LOH) and DNA-copy number abnormalities in clear cell renal cell carcinoma (cc-RCC) discovered by genome-wide single nucleotide polymorphism (SNP) arrays. Genomic DNA from tumor and normal tissue of 22 human cc-RCCs was analyzed on the Affymetrix GeneChip Human Mapping 10K Array. The array data were validated by quantitative polymerase chain reaction and immunohistochemistry. Reduced DNA copy numbers were detected on chromosomal arm 3p in 91%, on chromosome 9 in 32%, and on chromosomal arm 14q in 36% of the tumors. Gains were detected on chromosomal arm 5q in 45% and on chromosome 7 in 32% of the tumors. Copy number abnormalities were found not only in FHIT and VHL loci, known to be involved in renal carcinogenesis, but also in regions containing putative new tumor suppressor genes or oncogenes. In addition, microdeletions were detected on chromosomes 1 and 6 in genes with unknown impact on renal carcinogenesis. In validation experiments, abnormal protein expression of FOXP1 (on 3p) was found in 90% of tumors (concordance with SNP array data in 85%). As assessed by quantitative polymerase chain reaction, PARK2 and PACRG were down-regulated in 57% and 100%, respectively, and CSF1R was up-regulated in 69% of the cc-RCC cases (concordance with SNP array data in 57%, 33%, and 38%). Genome-wide SNP array analysis not only confirmed previously described large chromosomal aberrations but also detected novel microdeletions in genes potentially involved in tumor genesis of cc-RCC.

Figure 1

Graphical representation of chromosomal imbalances detected in genomes of patients with cc-RCC. Losses associated with LOH are indicated as red lines and green lines denote regions of copy number gains. Orange line points to a single occasion where copy number loss was not associated with LOH and blue lines indicate regions with LOH without copy number loss. The most frequent alterations are LOH due to hemizygous deletion on chromosomes/arms 3p, 9, and 14 and gains on 5q and 7. Patient IDs from left to right: 122, 126, 132, 135, 139, 154, 163, 167, 178, 182, 195, 201, 26, 28, 31, 44, 4, 65, 69, 7, 81, and 88.

Figure 2

Copy number and LOH profile of patient 154 in detail. (A) Copy number ratios in log₂ scale between the tumor DNA and unaffected DNA from the same patient. Each spot indicates a unique SNP locus corresponding to its genomic position aligned from 1pter (left) to Xqter (right). Each individual chromosome is coded in a different color. (B) SAS (=LOH) values for those SNP loci that were informative (i.e., biallelic) in the control DNA. Markers that were not informative (monoallelic) in the healthy control DNA are not shown, reducing the amount of data points by roughly two-thirds. In this case, losses in the copy number profile (on chromosomes 3p, 6p, and 8p) are always associated with LOH, so that a hypodiploid karyotype has to be assumed. Interestingly, the gain on chromosome 7 is not accompanied by a shift in the allele ratio. We interpret this constellation as a sign of a mixed population of cells having either two paternal and one maternal chromosome 7 and cells with two maternal and only one paternal chromosome 7.

Figure 3

Graphical representation of qPCR results. The mRNA expression of CSF1R was analyzed by qPCR in 16 matched pairs of malignant and nonmalignant tissues. A twofold up-regulation (solid line) was considered as threshold for altered expression in tumor samples. For one case (#154), no n-fold change could be calculated because the CSF1R expression level in the nonmalignant sample was below the detection limit. This sample pair belongs to the group with altered expression.

Figure 4

Expression of FOXP1 by immunohistochemistry. FOXP1 nuclear and cytoplasmic expression in normal renal tissue (A; original magnification, x20); nuclear FOXP1 expression in cc-RCC (B; original magnification, x20); lack of FOXP1 expression in cc-RCC (C; original magnification, x10); and abnormal concomitant membranous and nuclear FOXP1 expression in cc-RCC (D; original magnification, x20).