OS12.6 Intracranial GBM PDX Mutations Induced by Temozolomide Treatment

Abstract

Abstract BACKGROUND Temozolomide (TMZ) is routinely used in the post-surgical treatment of patients with newly diagnosed glioblastoma (GBM). Nearly all GBM recur following TMZ treatment, and there is an increasing body of data that detail TMZ-associated mutations in recurrent GBM. In vivo models of recurrent GBM are scarce in comparison to models of treatment-naïve GBM. MATERIAL AND METHODS Mice with intracranial treatment naïve GBM patient derived xenograft (PDX) were treated with radiotherapy (RT) only, TMZ only, or RT + TMZ. Tumors were monitored for recovery from treatment by bioluminescence imaging (BLI), and upon animal subject decline due to growing tumor, subjects were euthanized, with viable intracranial tumor dissected and used to establish subcutaneous tumor in new animal hosts that received no treatment. Subcutaneous tumors were sequentially passaged through two additional animal hosts. DNA was extracted from third passage subcutaneous tumors and subjected to full exome sequencing. PDX exome sequence data were examined for genes with nonsynonymous frameshift and nonsense variants (mutations). RESULTS Exome sequence analysis showed striking differences between PDX receiving RT only vs. TMZ with or without RT. Most notable is the substantially greater incidence of G-C to A-T transition mutations in TMZ-treated PDX, which are a signature of TMZ treatment. All TMZ-treated PDX also showed substantially more genes being mutated than PDX receiving RT only: one cycle RT only = 88; one cycle TMZ only = 339; one cycle of concurrent RT + TMZ = 531; one cycle of concurrent RT + TMZ followed by two additional cycles of TMZ only = 1502. The PDX subjected to multiple cycles of TMZ treatment prior to establishment as a subcutaneous tumor has shown stable resistance to TMZ treatment in subsequent experiments. Importantly, TMZ-treated PDX had mutations in LTBP4, MSH6, and PREX1 genes that are enriched in patient GBM following recurrence after initial TMZ treatment (Nat Genet 2016;48:768–76); these genes were not mutated in PDX receiving RT only. CONCLUSION Our study results support the following: 1) Intracranial GBM PDX treated with TMZ develop a global genomic treatment signature, as well as specific mutations that are observed in recurrent patient GBMs; 2) Mutations detected in TMZ-treated intracranial tumors are stable, persisting after repeated subcutaneous passages without further treatment; 3) Paired naïve and TMZ-treated GBM PDX will facilitate studies aimed at increasing our understanding of TMZ resistance, and help identify new therapies for more effective treatment of recurrent GBM.