To address the need for understanding ITH in CRC comprehensively and accurately, we used targeted deep next-generation sequencing of 799 genes to characterize the genetic profiles of four CRCs, followed by validation with ultra-high-depth amplicon sequencing. We also performed NanoString gene expression profiling and NanoString cancer copy number analysis.. Multi-region ultra-deep sequencing reveals early inter-mixing and variable levels of intratumoral heterogeneity in colorectal cancer

Background: 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 genes pathways in colorectal cancer (CRC). Methods: We performed multi-region, high-depth (384x on average) sequencing of 799 cancer-associated genes in 24 spatially separated primary tumor and non-malignant tissues from 4 treatment-naïve CRC patients. We then used ultra-deep sequencing (17,000x on average) to accurately verify 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 subclonal point mutations and determined the mutational timing and phylogenetic relationships among spatially separated sectors of each tumor. Results: Truncal mutations, those shared by all sectors in the tumor, affected well described driver genes such as APC, TP53, and KRAS. With sequencing at 17,000x, we found that mutations first detected at a sequencing depth of 384x 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. Conclusions: 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 subclonal mutations with ultra-deep sequencing, we traced the genomic histories of each tumor and relative timing of mutational events. We found evidence of early mixing, in which subclonal ancestral mutations inter-mixed across sectors before the acquisition of subsequent non-truncal mutations. Our observation is consistent with the “Big Bang model” of intra-tumoral spatial heterogeneity in CRC. Our findings also indicate that different CRC patients display markedly variable ITH, suggesting that each patient’s tumor possess a unique genomic history and spatial organization.