Extrachromosomal circular DNA orchestrates genome heterogeneity in urothelial bladder carcinoma

Rationale: Extrachromosomal circular DNA is a hallmark of cancer, but its role in shaping the genome heterogeneity of urothelial bladder carcinoma (UBC) remains poorly understood. Here, we comprehensively analyzed the features of extrachromosomal circular DNA in 80 UBC patients. Methods: We performed whole-genome/exome sequencing (WGS/WES), Circle-Seq, single-molecule real-time (SMRT) long-read sequencing of circular DNA, and RNA sequencing (RNA-Seq) on 80 pairs of tumor and AT samples. We used our newly developed circular DNA analysis software, Circle-Map++ to detect small extrachromosomal circular DNA from Circle-Seq data. Results: We observed a high load and significant heterogeneity of extrachromosomal circular DNAs in UBC, including numerous single-locus and complex chimeric circular DNAs originating from different chromosomes. This includes highly chimeric circular DNAs carrying seven oncogenes and circles from nine chromosomes. We also found that large tumor-specific extrachromosomal circular DNAs could influence genome-wide gene expression, and are detectable in time-matched urinary sediments. Additionally, we found that the extrachromosomal circular DNA correlates with hypermutation, copy number variation, oncogene amplification, and clinical outcome. Conclusions: Overall, our study provides a comprehensive extrachromosomal circular DNA map of UBC, along with valuable data resources and bioinformatics tools for future cancer and extrachromosomal circular DNA research.


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Figure S1.The experimental workflow of the CCGA-UBC study and the clinical characteristics of the patients (n = 80) (A) A total of 80 pairs of freshly snap-frozen tumor tissues and Adjacent Tumor Tissues (ATs) were collected from UBC patients.Hematoxylin-Eosin (HE) staining was performed to assess tumor purity and pathology.Sample filtering was performed based on the following criteria: 1) Tumor tissues with tumor purity above 80%; 2) Successful isolation of high molecular weight genomic DNA (HMW gDNA) and qualified RNA (RNA integrity number > 5) from both tumor and AT samples.

Figure S2 .
Figure S2.Focal amplification landscape of CCGA-UBC Samples (A) Summary of the definitions and functions of two main types of extrachromosomal circular DNA (ecDNA and eccDNA).
(B) Pie chart showing the percentages of tumors with different numbers of ecDNA species among the total 45 ecDNA + cases.(C) Genomic distribution of each amplicon class, including ecDNA, BFB, Heavilyrearranged, and Linear.(D) Oncogene profile and associated clinicopathologic features of patients with any type of focal amplification (carrying oncogenes) in the CCGA-UBC cohort (n = 46) and TCGA-BLCA cohort (n = 68).(E-F)AA-generated structural variant (SV) and breakpoint graph of examples of a simple cycle (E) and a complex multi-chromosomal circular amplification (F).

Figure S3 .
Figure S3.Validation of CCND1 amplification by Interphase FISH (A) AA-generated structural variant (SV) and breakpoint graph of four CCND1containing ecDNA.(B) Interphase FISH microscopy targeting the amplified CCND1 in four available formalin-fixed paraffin-embedded (FFPE) tumor samples.The green and red signals represent the CCND1 gene and Centromere 11 (CEN11) region, respectively.Scale bar, 10 μm.

Figure S4 .
Figure S4.Genome-scale map of small extrachromosomal circular DNAs (eccDNAs) in CCGA-UBC Samples (A) Circle-Seq method for mapping of eccDNAs.Paired AT-tumor samples were collected from 80 CCGA-UBC patients.Total high molecular weight genomic DNA (HMW gDNA) was isolated from tissues using a magnetic-based approach.The linear portions of HMW gDNA were hydrolyzed by Exonuclease V (Exo V).PCR was performed to validate the removal of linear DNA.The purified circular DNAs were amplified by phi29 polymerase.The amplified products were subjected to MGI PE150 sequencing (n = 80 pairs) and long-read SMPT sequencing (PacBio) (n = 9 pairs).The eccDNAs were detected from sequencing data by Circle-Map ++ and the Consensus eccDNA generation method (see methods).

Figure S5 .
Figure S5.Correlation analysis of eccDNA abundance and mRNA expression at the gene level (A) Ranked of mRNA expression, eccDNA abundance, and CNA value in CCGA-UBC-065 tumor sample.(B) Correlation analysis of eccDNA and mRNA (6,918 mRNA-eccDNA pairs) in tumors and adjacent tissues (ATs) using Spearman's correlation.(C) Pathways enriched in genes with significant eccDNA-mRNA correlation in ATs (left) and tumors (right).(D)Overlap of genes genes exhibiting significant changes in both gene expression levels and eccDNA abundance in tumors and ATs.

Figure S6 .
Figure S6.Correlation analysis of ecDNA with clustered somatic mutations and chromothripsis (A) Distances to the nearest breakpoints for BFB (Left), Heavily-Rearranged (Middle), and Linear (Right) events associated with kataegis mutations versus non-clustered mutations.(B) Mutational signatures associated with kataegis events.(C) Number of mutations for each mutational signature (Est_Counts) and their normalized contributions across samples (Fraction).(D) Mutational signatures of kataegis within 10kb of ecDNA breakpoints (E) Co-occurrence of chromothripsis with focal amplification.Sample level analysis of chromothripsis distribution across different amplification samples and locus-level overlap between chromothripsis and each type of amplicon,

Figure S8 .
Figure S8.Multivariate analysis of associations between EPM and clinical variables and prognosis.