Molecular genetic characterization of retinoblastoma tumors lacking RB1 mutations

Retinoblastoma is a rare childhood cancer of the retina and is the most common intraocular tumor in children. Classically, retinoblastoma results from biallelic loss of the RB1 tumor suppressor gene. As with other cancer types, dysregulation of a single gene alone is not considered sufficient for complete transformation to malignancy. Frequent regions of genetic alteration harbouring additional genes, implicated in retinoblastoma oncogenesis and progression, include chromosomes 1q, 2p, 6p, 13q and 16q. Sensitive molecular genetic screening techniques are capable of identifying RB1 mutations in 98% of unilateral retinoblastoma tumors. The remaining 2% harbour no identifiable RB1 inactivating alterations, and therefore molecular interrogation of these cases would likely reveal alternative genetic events driving retinoblastoma tumorigenesis in the absence of RB1 inactivation. Towards this objective, in this thesis work, I describe genetic alterations identified by tiling path array comparative genomic hybridization in a rare sample set composed of 23 RB1⁺/⁺ tumors. In addition to gene disruption by copy number alteration, mechanisms of gene disruption resulting in no overall change in copy number or change in copy number with allelic imbalance were also investigated utilizing genome-wide SNP array analysis on five of the RB1⁺/⁺ tumors. The most striking recurrent genetic alteration identified in retinoblastoma tumors lacking RB1 inactivating mutations, was focal high-level MYCN amplification, which occurred at a frequency of approximately 48%. The MYCN amplified RB1⁺/⁺ tumors also exhibited a statistically significant lower proportion of their genome affected by genomic instability when compared with the RB1 ⁻/⁻ tumors. In a subset of five matched tumor and blood normal samples the occurrence of copy neutral LOH was explored, although none was observed. Allele specific copy number analysis identified instances of allelic imbalance, including all five MYCN amplifications. Amplification of MYCN may represent a rare and novel alternate mechanism of retinoblastoma tumorigenesis. This work provides insight into the role of mutational events driving tumorigenesis in retinoblastoma in the absence of RB1 inactivation.Medicine, Faculty ofPathology and Laboratory Medicine, Department ofGraduat