Figure 4. Extrachromosomal DNA marks subclones driving tumor progression in patient tumors and derived model systems.

A. Establishing neurosphere cultures and PDX models from a paired primary/recurrent GBM. B. DNA copy number analysis shows co-amplification of EGFR (chr7)/CDK4 (chr 12) is detected in primary GBM HF3016 which is sustained in both neurosphere and xenografts derived from this primary tumor, as well as the recurrent GBM HF3177, and the neurosphere/xenografts thereof. The HF3016 primary tumor is not MYC amplified. The HF3016 neurosphere, as well as all HF3177 samples, show focal MYC amplification. C. Representative FISH images from 50 metaphase and 100 interphase nuclei for MYC (red) and Ch8 marker (green) show that a small fraction (2%) of the cells in HF3016 tumor presents MYC amplification, while 100% of nuclei in the remaining samples present MYC amplification, which is clearly extrachromosomal (white arrows) in the metaphase spreads (NS). D. Clonal tracing of a pair of primary-recurrent GBM, their matching neurospheres, and xenografts. Each line represents a group of mutations computationally inferred to reflect a subclone. E. Starting in the neurosphere of the primary tumor, a complex structural variant is identified that connects the CDK4 locus to the EGFR locus. The MYC locus is not part of this variant. The EGFR/CDK4 variant is detected in HF3016 PDXs as well as all HF3177 samples. F. EGFR (green) and CDK4 (red), detected by FISH, are amplified in 100% of nuclei for every sample from this patient, with identical copy numbers in each nucleus (bottom of the panels). Overlapping dots show that EGFR/CDK4 co-localize (white arrows) and metaphase FISH (NS) shows extrachromosomal co-amplification in the same double minute (inserts). Images are representative of 50 metaphase and 100 interphase nuclei per group. Scale bars, 3 μm.