Abstract 1140: Intratumoral mutual exclusivity of dual amplified receptor tyrosine kinase genes in glioblastoma
Authors: Little Suzie , Jury Alexa , Bax Dorine , Doey Lawrence , Al-Sarraj Safa , Jurgensmeier Juliane , Jones Chris 
Published: August 2012

Abstract
Cancer heterogeneity presents a major barrier for the development of effective treatment strategies aimed at combating the disease. Within glioblastoma, this intratumoral heterogeneity may refer to the concurrent observation of multiple cellular morphologies, differential patterns of vascular proliferation, discrete transcript and/or protein expression patterns, and genetic variegation. DNA copy number profiling has demonstrated that multiple amplifications may frequently be found in the same glioblastoma specimens. Although tumour clonality would imply that these events would be present in all neoplastic cells, we and others have previously noted through detailed FISH/CISH experiments on pathological specimens that not all cells harbour individual amplification events. In order to explore these concepts more thoroughly, tissue microarrays comprising 342 high grade glioma samples were screened by FISH for cells amplified for genes encoding either EGFR or PDGFRA. A total of 18 cases (8%) contained both gene amplifications in at least one cell of the TMA core. We utilised two-colour FISH with differential labelling of probes specific to EGFR and PDGFRA on whole sections from these samples, and assessed the relative copy numbers per cell of each gene. The most common pattern comprised mutual exclusivity of gene amplification within adjacent glioblastoma cells. To quantify the extent to which these patterns were reflected across the tumour, we carefully mapped the relative copy numbers of EGFR and PDGFRA in cells from throughout the pathology specimen of all dual-amplified glioblastoma cases. By counting signals in 41,997 cells from 190 distinct loci across 17 samples, we noted a startling heterogeneity of amplification patterns both across and within tumour specimens, with approximately three-quarters of cells harbouring amplification of only one or other of the genes. In only one case did the single-cell ‘co-amplification’ model represent more than 15% of the sample as a whole. Within an individual sample, cells harbouring one, both, or neither amplification could be found in highly variable frequencies, either presenting as a mosaic of genetically distinct cells, or forming foci where one event would strongly predominate. We observed that these PDGFRA-restricted cells tended to be present close to endothelial cells, whilst poorly vascularised areas were largely the domain of EGFR-amplified tumour cells. EGFR amplification was significantly linked with areas of glioblastoma containing large numbers of giant cells and/or small cells, whereas all high frequency PDGFRA-amplified cells were found in areas of fibrillary histology. These data have profound implications for designing efficacious therapeutic regimens, as the relative contributions of cell populations harbouring one or other gene amplification to disease propagation, and the implications for targeted therapies, are not known.
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 1140. doi:1538-7445.AM2012-1140