Abstract 3101: Transition from extrachromosomal to intrachromosomal amplification in glioblastoma cells

Abstract

Abstract Gene amplification is one of the genomic alterations found in cancer cells by which oncogenes may be over-expressed. Amplified sequences may be found within homogeneously staining regions of the chromosomes (hsr) or on autonomously replicating circular and acentric extrachromosomal DNA molecules, named double minutes (dmins). Integration of dmins is a recognized pathway for hsr formation, but the molecular mechanisms involved in this process are still poorly understood. We analyzed three xenografted glioblastomas with dmins at early passages in mice that evolved in hsr at late passages. Using various approaches (FISH, PCR, quantitative-PCR, chromosome walking, DNA sequencing, genome-wide SNP analysis), we characterized the events leading to dmin formation, evolution and reintegration, and determined the structure of the integration sites. In all three glioblastomas, dmins constituted by the fusion of several syntenic or non-syntenic DNA fragments formed upon chromosome fragmentation were present at early passages. The fragments were fused by a non-homologous end-joining (NHEJ) mechanism. Later, a new type of dmin was formed by the fusion of fragments from both the initial dmin and other chromosomes, suggesting a second round of extensive DNA fragmentation. The new type of dmin then gave rise to an hsr. It is not known whether the new dmin was amplified extrachromosomally before reintegration or if one molecule was integrated, then amplified in the chromosome by a mechanism that did not involved breakage-fusion-bridge cycles since the target chromosome was not rearranged outside of the integration site. The integration sites were characterized at the nucleotide level. No preferential integration sequence was found. No significant homologies existed between extrachromosomal and intrachromosomal sequences at the integration sites. The structure of the integration site may be simple, with a single small deletion, or very complex, exhibiting deletions, duplication and insertion of fragments coming from other regions of the genome. Fusions between fragments were mainly driven by NHEJ. Current data allow us to propose a mechanism of hsr formation through integration of extrachromosomal DNA molecules. At the best of our knowledge, it is the first reported molecular characterization of hsr insertion sites. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3101. doi:1538-7445.AM2012-3101