Abstract
Intratumoural heterogeneity underlies tumour escape from molecularly targeted therapy in glioblastoma. A cell-based model preserving the evolving molecular profiles of a tumour during treatment is key to understanding the recurrence mechanisms and development of strategies to overcome resistance. In this study, we established a matched pair of glioblastoma stem-like cell (GSC) cultures from patient glioblastoma samples before and after epidermal growth factor receptor (EGFR)-targeted therapy. A patient with recurrent glioblastoma (MGG70R) harboring focal, high-level EGFR amplification received the irreversible EGFR tyrosine kinase inhibitor dacomitinib. The tumour that subsequently recurred (MGG70RR) showed diploid EGFR, suggesting inhibitor-mediated elimination of EGFR-amplified tumour cells and propagation of EGFR non-amplified cell subpopulations. The MGG70R-GSC line established from MGG70R formed xenografts retaining EGFR amplification and EGFR overexpression, while MGG70RR-GSC established from MGG70RR generated tumours that lacked EGFR amplification and EGFR overexpression. MGG70R-GSC-derived intracranial xenografts were more proliferative than MGG70RR-GSC xenografts, which had upregulated mesenchymal markers, mirroring the pathological observation in the corresponding patient tumours. In vitro MGG70R-GSC was more sensitive to EGFR inhibitors than MGG70RR-GSC. Thus, these molecularly distinct GSC lines recapitulated the subpopulation alteration that occurred during glioblastoma evasion of targeted therapy, and offer a valuable model facilitating therapeutic development for recurrent glioblastoma.
Highlights
Despite the standard treatment with resection, radiotherapy, and the alkylating agent temozolomide[1], glioblastoma harbors a poor prognosis and remains a fatal disease for the vast majority of cases
Immunohistochemical analysis showed that MGG70R had diffuse and intense immunopositivity for epidermal growth factor receptor (EGFR) and its activated form phospho-EGFR, a very similar staining pattern to that observed in the original tumour MGG70 (Fig. 2, Supplementary Fig. S1)
Fluorescence in situ hybridization (FISH) analysis showed focal EGFR amplification in the newly diagnosed tumour MGG70, which was retained at a higher level in the recurrent MGG70R specimen (Fig. 3), suggesting that the treatment with radiotherapy and temozolomide did not preferentially target cell populations harboring amplified EGFR
Summary
Despite the standard treatment with resection, radiotherapy, and the alkylating agent temozolomide[1], glioblastoma harbors a poor prognosis and remains a fatal disease for the vast majority of cases. Patient accrual in these two trials was restricted to those with EGFR gene amplification in archival tumour specimens, with an expectation of their better response to PF-002998049. The latter phase II trial (NCT01520870) reported a limited activity of the drug in recurrent glioblastoma with EGFR amplification, a minor fraction of patients, 4 of 49 (8.2%), had durable ( > 6 months) response[10]. Possible escape mechanisms include intratumoural heterogeneity[11,12,13], loss of target gene expression and activation of redundant signaling pathways[14,15] Elucidating these resistance mechanisms in more detail is critical for future studies of second-generation molecularly targeted agents. We established GSC neurospheres from patient tumour samples harvested before and after treatment with an EGFR-targeted agent, and analysed the molecular and biological characteristics that the GSC and patient tumour specimens exhibited pre- and post-treatment with this targeted drug
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