Abstract 3881: In vivo and in vitro characterization of genomic diversity and clonal evolution in glioblastoma
Authors: deCarvalho Ana C. , Kim Hoon , Poisson Laila , Verhaak Roel , Mikkelsen Tom 
Published: August 2015

Abstract
Introduction
Glioblastoma (GBM) is the most prevalent and aggressive primary central nervous system tumor. The molecular heterogeneity among and within GBM tumors may contribute to therapy failure. Comprehensive panels of well characterized patient-derived xenograft models for pre-clinical studies is critical to development of novel GBM therapies.
Methods
We established neurosphere models (NSM) from twelve freshly resected primary and one recurrent GBM and intracranially implanted these in immunocompromised mice. Exome, low pass whole genome and whole transcriptome sequencing was performed on tumors, low passage NSM and macrodissected xenografts in biological triplicates (total nr of samples: 65).
Results
GBM transcriptional subtypes were conserved between tumor samples and matched NSM in 42% of cases, suggesting a role for the tumor microenvironment in expression classification. Filtering of mouse sequence reads from xenograft RNAseq data allowed us to limit subtype classification to the tumor cell only transcriptome and as a result, expression subtype was consistent in 83% of NSM and derived xenografts. The majority (&gt;95%) of driver gene mutations and copy number changes were preserved across all five instances of each parental tumor. Of all somatic mutations detected in parental tumor samples, 80% could be recovered in the NSM after correcting for LOH status and base coverage. Surprisingly, the percentage of recovered tumor sample mutations increased to 85% in xenografts which was robust in validation and suggested subclonal selection in vitro, but not elimination of the subclones. The microenvironment provided by NSM culture medium and nude mice cranium may provide variable proliferative advantages resulting in specific tumor subclones to become dominant. Homozygous deletions and fusion transcripts found in tumor samples and xenografts but not NSM corroborated these observations. While xenografts retained a higher number of tumor sample mutations, a median number of 14 mutations were acquired relative to NSM suggesting a more susceptible environment to novel somatic events. Biological xenograft triplicates showed remarkable overlap in mutational content, suggesting that clonal evolution of NSM in vivo is not stochastic but a controlled Darwinian process.
To determine whether serial passaging of NSM affects the heterogeneity, we performed genomic profiling of four matching late passage NSM and found that these retained all mutations detected in their early passage progenitors, but acquired as many as 50 novel of mutations during the culturing process.
Discussion
Our study shows that neurosphere cultures and first generation xenografts closely mimic their parental tumor. NSM showed a remarkable heterogeneity and selective pressures from the tumor microenviroment resulted in reproducible subclonal evolution. Our findings provide critical new insights in our understanding of tumor model development.
Citation Format: Ana C. deCarvalho, Hoon Kim, Laila Poisson, Roel Verhaak, Tom Mikkelsen. In vivo and in vitro characterization of genomic diversity and clonal evolution in glioblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3881. doi:10.1158/1538-7445.AM2015-3881