Multiregion ultra‐deep sequencing reveals early intermixing and variable levels of intratumoral heterogeneity in colorectal cancer

Intratumor heterogeneity (ITH) contributes to cancer progression and chemoresistance. We sought to comprehensively describe ITH of somatic mutations, copy number, and transcriptomic alterations involving clinically and biologically relevant gene pathways in colorectal cancer (CRC). We performed multiregion, high‐depth (384× on average) sequencing of 799 cancer‐associated genes in 24 spatially separated primary tumor and nonmalignant tissues from four treatment‐naïve CRC patients. We then used ultra‐deep sequencing (17 075× on average) to accurately verify the presence or absence of identified somatic mutations in each sector. We also digitally measured gene expression and copy number alterations using NanoString assays. We identified the subclonal point mutations and determined the mutational timing and phylogenetic relationships among spatially separated sectors of each tumor. Truncal mutations, those shared by all sectors in the tumor, affected the well‐described driver genes such as APC, TP53, and KRAS. With sequencing at 17 075×, we found that mutations first detected at a sequencing depth of 384× were in fact more widely shared among sectors than originally assessed. Interestingly, ultra‐deep sequencing also revealed some mutations that were present in all spatially dispersed sectors, but at subclonal levels. Ultra‐high‐depth validation sequencing, copy number analysis, and gene expression profiling provided a comprehensive and accurate genomic landscape of spatial heterogeneity in CRC. Ultra‐deep sequencing allowed more sensitive detection of somatic mutations and a more accurate assessment of ITH. By detecting the subclonal mutations with ultra‐deep sequencing, we traced the genomic histories of each tumor and the relative timing of mutational events. We found evidence of early mixing, in which the subclonal ancestral mutations intermixed across the sectors before the acquisition of subsequent nontruncal mutations. Our findings also indicate that different CRC patients display markedly variable ITH, suggesting that each patient's tumor possesses a unique genomic history and spatial organization.