Resolving clonal evolution and selection of extrachromosomal DNA at single-cell resolution

BACKGROUND: Genomic alterations are a hallmark of cancer, and extrachromosomal DNA (ecDNA) has emerged as a key source of oncogene selection, tumor growth, and drug resistance. The intratumor heterogeneity and clonal selection of ecDNA is, however, poorly understood. RESULTS: In this study, we pursue a computational approach that leverages allelic imbalance and outlier expression from standard single-cell RNA sequencing (scRNA-seq) to deconvolve the tumor heterogeneity of ecDNA at the single-cell level (ecSingle). Using this approach, we identify oncogene-carrying ecDNAs in tumor samples at the single-cell level, which we validate using genome sequencing. Moreover, we show the superiority of using single-molecule long-read sequencing in resolving ecDNA. ecDNAs displayed extensive intratumor heterogeneity, including subclonal oncogene-carrying ecDNA in primary tumor cells that segregate with distinct transcriptional cell states. Importantly, we show that a rare ecDNA+ clone in the primary tumor can expand to form dominant clones in relapse tumors. CONCLUSIONS: Our study introduces a novel approach to studying ecDNA at the single-cell level, enabling both clonal evolution and transcription cell state analysis. We apply this approach to cancer samples to gain deeper insights into the role of ecDNA in intratumor heterogeneity and cellular plasticity.