Abstract 2916: The clonal evolution of glioblastoma
Authors: Goldstein Jennifer Brooke , Ezhilarasan Ravesanker , Jaffari Mona , Carugo Alessandro , Draetta Giulio , Verhaak Roeland , Seth Sahil , Sulman Erik , Futreal Phillip Andrew 
Published: July 2017

Background: Glioblastoma (GBM) is an aggressive cancer that often recurs despite multimodal therapy. Median survival is 12-15 months. Genomic profiling studies have shown marked tumor heterogeneity with distinct mutations among treated and untreated samples. The Cancer Genome Atlas (TCGA), revealed that mutations in TP53, CDKN2A, PTEN, EGFR and NF1 are predominant in untreated tumors. Of 19 patients who progressed on therapy, seven samples were hypermutated with mutations in mismatch repair proteins, mainly MSH6. We proposed to use the Cellecta Lentiviral-based tagging library, a novel model system to assess the role of intratumor heterogeneity (ITH) in the presumptive tumor-initiating fraction of primary GBM and in the development of resistance to temozolomide (TMZ). From a pool of 30,000,000 barcodes, the system enables the genetic integration of a unique barcode sequence into each cell. Each barcode can be quantitatively tracked via next-generation sequencing, allowing for dynamic monitoring of the subclonal architecture.

Methods: GSC 272 and 627 (sensitive and resistant to TMZ) were derived from core biopsies of GBM patients. Cells were transduced with a lentiviral vector containing luciferase. Cells were expanded and infected with the Cellecta Lentiviral library. Multiplicity of infection was determined. Transduced cells were injected into B6.Cg-Foxn1nu/J mice brains using a guide screw system. Half of the mice were treated with TMZ via gavage at two weeks. Brain tumors were removed and total DNA was extracted. Barcode inserts were amplified and the Illumina Sequencing platform was used for barcode quantification. Additionally, whole exome sequencing was performed to assess mutation status.

Results: Confirmation of luciferase signal has been performed using a Luciferase Reporter Assay. Multiplicity of infection was found to be 0.15 and 1 for GSC 6-27 and 272 respectively. Initial methylation studies showed GSC 272 to have methylation of MGMT while GSC 627 did not. There was no difference in OS in the TMZ Resistant (GSC 627) untreated vs. treated cohort (with a median OS of 55 days in both groups (N= 23 vs 25 respectively, P= 0.563). TMZ Sensitive (GSC 272) untreated mice did not survive as long as the treated cohort (N= 24 vs. 25, Median OS 43 vs. 206 days, P= 4.61e-09). Barcode and whole exome sequencing results is currently being analyzed.

Conclusions: The Cellecta lentiviral tagging system is an innovative way to track ITH and clonal evolution in glioblastoma orthotopic models. We hope to discover novel insights into TMZ treatment response and resistance.

Citation Format: Jennifer Brooke Goldstein, Ravesanker Ezhilarasan, Mona Jaffari, Alessandro Carugo, Giulio Draetta, Roeland Verhaak, Sahil Seth, Erik Sulman, Phillip Andrew Futreal. The clonal evolution of glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2916. doi:10.1158/1538-7445.AM2017-2916