Journal: Cancer Discovery

Article Title: Extrachromosomal DNA–Driven Oncogene Spatial Heterogeneity and Evolution in Glioblastoma

doi: 10.1158/2159-8290.CD-24-1555

Figure Lengend Snippet: Diversity and high copy numbers of ecDNA in IDH wt GBMs. A, Focal copy-number amplifications across the GB-UK and PCAWG cohorts, categorized by amplicon type [ecDNA, breakage–fusion–bridge (BFB) cycle, linear]. Only tumors containing focal amplifications are shown. Oncogenes with the same colored box indicate they exist on the same ecDNA molecule. The horizontal line between patients A61 and A3 (GB-UK) and patients SP27603 and SP23622 (PCAWG) separates patient samples with (above) or without (below) ecDNA, detected by WGS, DNA FISH, or nascent RNAscope. Oncogenes tested with DNA FISH and/or nascent RNAscope, EGFR , PDGFRA , and CDK4 , are separated to the left of the vertical dashed line. B, Median ecDNA copy number vs. number of ecDNA-amplified genes for GB-UK and PCAWG cohorts (Spearman correlation). C, Oncogenes contained within ecDNA in both GB-UK and PCAWG cohorts ranked by maximum copy number. The number of samples with each oncogene as ecDNA in the GB-UK cohort is described in brackets. Only copy numbers detected by WGS are shown here. D, Mean copy number of linear focal amplifications vs. ecDNA in GB-UK. E, Mean copy number of non-oncogenes compared with oncogenes amplified on ecDNA in GB-UK. F, Complexity score of linear focal amplifications vs. ecDNA in GB-UK. G, Number of uni- and multichromosomal ecDNAs in GB-UK and PCAWG cohorts. H, Distribution of ecDNA lengths in GB-UK and PCAWG cohorts. I, Median ecDNA copy number vs. length of intergenic DNA for EGFR -ecDNA in GB-UK and PCAWG cohorts (Spearman correlation).

Article Snippet: Gene-specific probes containing chromosome-specific centromere enumeration probes against PDGFRA (Empire Genomics), EGFR (Vysis/Abbott), and Myc (Vysis/Abbott) were applied directly to the tissue sections with the coverslip being sealed with rubber solution glue.

Techniques: Amplification, RNAscope

Journal: Cancer Discovery

Article Title: Extrachromosomal DNA–Driven Oncogene Spatial Heterogeneity and Evolution in Glioblastoma

doi: 10.1158/2159-8290.CD-24-1555

Figure Lengend Snippet: SPECIES spatial modeling of ecDNA evolution. A and B, DNA FISH staining of two representative GBM samples from the GB-UK cohort, revealing ( A ) EGFR - and ( B ) PDGFRA -ecDNA. EGFR -ecDNA seem to congregate into “hubs” (white arrows). Single-cell ecDNA copy-number distributions are derived from DNA FISH images using unbiased image analysis. C, (i) SPECIES is initiated with a single tumor cell containing k copies of ecDNA. (ii) Model of ecDNA selection. Cells carrying one or more copies of ecDNA divide at a rate s faster than cells lacking ecDNA. (iii) Cells push neighbors within a radius q on the lattice to make space required for cell division. D, Spatial computational model of ecDNA-driven tumors. ABC was used to infer optimal model parameters for the initial number of ecDNA, k ; ecDNA-conferred selection, s ; and cell pushing strength, q , for a given set of patient tumor measurements. E and F, Example output of the ABC model fitting algorithm using ( E ) 2 and ( F ) 3 spatial regions. (Left to right) Inferred k , s , and q and model best-fit distributions for each tumor region. The sum of the Wasserstein distance between patient and simulated distributions for each tumor region, representing closeness of fit, is denoted by σ. G, Comparison of inferred k , s , and q when analyzing 2 (core and margin) vs. 3 (core, margin, and leading edge) spatial regions. H, Summary of inferred model parameters for all sampled human GBM tumors. The highlighted color represents ecDNA-amplified oncogenes ( EGFR , PDGFRA , or CDK4 ) in each tumor. I, Inferred model parameters for all patient tumors (excluding the tumor of patient A5, which contained separate EGFR- and PDGFRA -ecDNA species), stratified by ecDNA-amplified oncogene ( EGFR or PDGFRA ). ( D, created using BioRender assets. https://BioRender.com/7f2pume )

Article Snippet: Gene-specific probes containing chromosome-specific centromere enumeration probes against PDGFRA (Empire Genomics), EGFR (Vysis/Abbott), and Myc (Vysis/Abbott) were applied directly to the tissue sections with the coverslip being sealed with rubber solution glue.

Techniques: Staining, Derivative Assay, Selection, Comparison, Amplification

Journal: Cancer Discovery

Article Title: Extrachromosomal DNA–Driven Oncogene Spatial Heterogeneity and Evolution in Glioblastoma

doi: 10.1158/2159-8290.CD-24-1555

Figure Lengend Snippet: Simulating multi-ecDNA dynamics in vivo. A, DNA FISH and nascent RNAscope images from the tumor of patient A5, showing the coexistence of both EGFR -ecDNA and PDGFRA -ecDNA. B, Circular structures of three distinct oncogenic ecDNAs detected in the tumor of patient A5. C, GBMs in the GB-UK and PCAWG cohorts containing more than one oncogenic species and their respective copy numbers. D, In a multiple-ecDNA application of SPECIES, the clone-initiating cell begins with k 1 and k 2 copies of ecDNA 1 and ecDNA 2, respectively. E, Representation of the impacts of ecDNA cosegregation and coselection on the number of inherited ecDNA copies during cell division. Cosegregation drives correlated inheritance of both ecDNA types, whereas coselection favors tumor cells carrying at least one copy of each type. F, Application of SPECIES to simulate two separate ecDNA species. Simulated tumors were initialized with a single cell, carrying k 1 and k 2 copies of each ecDNA species. Representative model images show examples of low and high cosegregation and coselection. G and H, Parameter inference summary for the tumor of patient A5, for which we measured both the EGFR -ecDNA and PDGFRA -ecDNA copy-number distributions using DNA FISH, using multiple-ecDNA SPECIES. Parameters s p and s m represent selection coefficients for tumor cells with 1 (pure) or 2 (mixed) ecDNA species, respectively. I, Comparison of inferred k and k 1 values for all patient tumors confirmed by WGS to harbor two or more ecDNA species. J, The presence of ecDNA amplifications may be used to aid stratification, given that oncogenes on ecDNA have an inherent resistance mechanism through the ability of ecDNA to dynamically adjust copy number in response to targeted agents. Earlier monitoring and intervention are recommended for those patient tumors with ecDNA that will receive targeted therapies.

Article Snippet: Gene-specific probes containing chromosome-specific centromere enumeration probes against PDGFRA (Empire Genomics), EGFR (Vysis/Abbott), and Myc (Vysis/Abbott) were applied directly to the tissue sections with the coverslip being sealed with rubber solution glue.

Techniques: In Vivo, RNAscope, Selection, Comparison