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

Abstract

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.

Individualised multiplexed circulating tumour DNA assays for monitoring of tumour presence in patients after colorectal cancer surgery

“…As already noted above, it has been shown that, at least in CRC, there is about 80% concordance between somatic mutations found in primary and metastatic tumours1011. Also, recent studies by us – using targeted capture sequencing with the same 799-gene panel used here – as well as others, have further demonstrated that there is a high level of concordance of mutations present across different sectors of patients’ primary colorectal tumours212223. As such, tumour heterogeneity should not significantly affect our assays.…”

A population genetics perspective on the determinants of intra-tumor heterogeneity

“…Computational modeling of spatial tumor growth and statistical inference on the genomic data revealed that most detectable ITH occurs early during tumor growth, long before the lesion is clinically evident. These findings were subsequently corroborated by reports that subclonal alterations originate early in colorectal adenomas/carcinomas [45–48]. Hence, ITH is already present in early lesions and continues to accrue during progression.…”

“…Subsequent studies have corroborated aspects of ‘Big Bang’ dynamics [46, 47, 247], suggesting that they may be relatively common in colorectal tumors. Others have considered more strict models of neutrality.…”

Spatial structure governs the mode of tumour evolution

“…A primary goal of modern cancer research is to characterize this evolutionary process, so as to enable precise, patient-specific prognostic forecasts and to optimize targeted therapy regimens.Much progress has been made in revealing the evolutionary features of particular cancers, yet these findings raise as many questions as they answer. Why do different tumour types exhibit different modes of evolution [1,3,4,5,6]? What conditions sustain the frequently observed pattern of branching evolution, in which several clones diverge and evolve in parallel [1,7]?…”