High-resolution DNA copy number and gene expression analyses distinguish chromophobe renal cell carcinomas and renal oncocytomas

Abstract

Background: The diagnosis of benign renal oncocytomas (RO) and chromophobe renal cell carcinomas (RCC) based on their morphology remains uncertain in several cases.

Methods: We have applied Affymetrix GeneChip Mapping 250 K NspI high-density oligoarrays to identify small genomic alterations, which may occur beyond the specific losses of entire chromosomes, and also Affymetrix GeneChip HG-U133 Plus2.0 oligoarrays for gene expression profiling.

Results: By analysing of DNA extracted from 30 chRCCs and 42 ROs, we have confirmed the high specificity of monosomies of chromosomes 1, 2, 6, 10, 13, 17 and 21 in 70-93% of the chRCCs, while ROs displayed loss of chromosome 1 and 14 in 24% and 5% of the cases, respectively. We demonstrated that chromosomal gene expression biases might correlate with chromosomal abnormalities found in chromophobe RCCs and ROs. The vast majority genes downregulated in chromophobe RCC were mapped to chromosomes 2, 6, 10, 13 and 17. However, most of the genes overexpressed in chromophobe RCCs were located to chromosomes without any copy number changes indicating a transcriptional regulation as a main event.

Conclusion: The SNP-array analysis failed to detect recurrent small deletions, which may mark loci of genes involved in the tumor development. However, we have identified loss of chromosome 2, 10, 13, 17 and 21 as discriminating alteration between chromophobe RCCs and ROs. Therefore, detection of these chromosomal changes can be used for the accurate diagnosis in routine histology.

Figure 1

Copy number alteration in a chromophobe RCC and RO. (A) Representative genome view of copy number alterations of chromosomes 1, 2, 6, 10, 13, 17 and 21 in a chromophobe RCC and of chromosome 1 as a single genomic change in a RO. (B) The dark green bars represent heterozygous SNP calls in tumors. (C) The yellow bar marks the copy number data in a color-coded HMM model (pink: copy number 3, yellow: copy number 2, blue: copy number 1).

Figure 2

Summary of genomic imbalances in 30 chRCCs and 42 ROs obtained with 250 K SNP array analysis. Green lines on the right of ideograms indicate losses, whereas red lines represent gains. Notice the smaller overlapping deletions at chromosome 11p and 12p in two and three cases of chRCC, respectively. Notice the loss of chromosome 1 and gain of the X chromosome in RO. Loss at the PAR1 region indicates the loss of the Y chromosome.

Figure 3

Homozygous deletion and gene expression. (A) A case of chromophobe RCC with a homozygous deletion at 21q21.3-q22.11 as revealed by SNP-based analysis. (B) Corresponding to the loss of both copies of the gene sequences the lack of expression of CLDN8 gene as obtained by quantitative real-time PCR.

Figure 4

Frequency of loss of entire chromosomes in chRCCs and ROs. Chromosomal changes occurring exclusively in chromophobe RCCs are marked by star.

Figure 5

Hierarchical clustering of gene expression profiles and comparative genomic microarray analysis of RO and chRCC. (A) Genomic regions showing a significant number of up- and down-regulated genes are red and green, respectively. (B) Copy number changes of chromophobe RCCs and ROs are marked by green (loss) and red (gain). Generally, loss of chromosomes 1, 2, 6, 10, 13, 17 and 21 is associated with the downregulation of genes. However, genes at chromosomes 16, 19, 20 and 22 found to be overexpressed in spite of the normal diploid copy number of these chromosomes.