Identification of genes involved in progression of retinoblastoma
Many retinoblastomas (RBs) show genomic alterations in addition to mutational loss of both normal RB1 alleles. The most frequent of these changes are gains on chromosomes 1q and 6p and losses on 16q. To identify the genes targeted by gains on chromosome 1q we used quantitative-multiplex PCR and array-CGH to determine DNA copy number changes in 76 primary tumours and 6 RB cell lines. In addition, in 21 of these tumours gene expression was analyzed by cDNA microarray hybridization. We have used microsatellite analysis and pyrosequencing to further localise the region of allele loss of heterozygosity on chromosome 16. We have also set up a specific methylation assay for the CDH11 gene located on 16p22 that was previously considered to be the target of loss on 16. The results we obtained were compared with expression of the corresponding genes. A novel region of amplification on the short arm of chromosome 1 was determined with array-CGH, and with a combination of genomic results and expression data allowed us to analyse the possibility of a novel oncogene involved in progression of retinoblastoma. Increased copy numbers of loci on chromosome 1q were present in 34 (45%) primary tumours and in all 6 cell lines. Two regions of gain emerged, one in 1q32 and another in 1q21. Tumours with 1q gains showed higher RNA expression of several genes in these two regions. The clinical manifestation of tumours with and without gains was similar with regard to many aspects including size, necrosis and calcification. However, the distribution of age at diagnosis was remarkably distinct with earlier diagnosis in tumours without gains. This suggests that these tumours are either initiated earlier or grow faster than tumours with gains. This association with clinical manifestation indicates that gains on 1q are significant for the biology of retinoblastoma. The genes on 1q with copy number gains and overexpression here are candidates that need to be tested for their individual contribution to the progression of retinoblastoma. The targets of loss on chromosome 16 are not clearly identified, but our results suggest that the CDH11 gene is probably not the tumour suppressor gene involved in progression of retinoblastoma that was proposed before. The results we obtained with our analyses on chromosome 1p suggest a role for MYCL1 in retinoblastoma.