LAUSR

Introduction Colorectal cancer progresses in a multi-step manner with adenoma being the most well-known precursor lesion. Malignant polyps, which are adenomas containing a focus of adenocarcinoma, are suitable models to study the evolution of colorectal cancer progression. Although the mutations that accrue during the adenoma-to-carcinoma transition have been previously described, the evolutionary dynamics of copy number alterations (CNAs) involved in this malignant transformation have not been fully understood. Material and methods Here, we performed low-pass whole genome sequencing and targeted deep sequencing of the benign and malignant portion of 23 cases. To validate subclonal copy number alterations we performed single-cell FISH analysis using six probes on chromosomes 5, 7, 8, 13, 18, and 20. We applied FISHtrees to reconstruct the phylogenetic tree of 200 single cells from the adenoma and 200 single cells from the adenocarcinoma portion. Results and discussions By inferring a consensus phylogenetic tree from the same lesion, we deciphered a pattern of branched evolution in all samples evaluated. Analysis of FISH data showed that the levels of genomic heterogeneity were highest in malignant polyps compared to non-progressed adenomas, with the adenoma component having a lower degree of chromosomal instability than the adenocarcinoma. Nevertheless, we observed different patterns of clonal evolution including neutral dynamics and punctuated evolution. We estimated which genomic SCNA showed the greatest allelic expansion comparing the carcinoma to the adenoma and defined this expansion as the fitness gain for the alteration. Genomic gains affecting chromosomes 7, 13, and 20 showed the highest fitness values. Interestingly, whole genome duplication, confirmed by image cytometry, appeared to be a decisive step in the transition from adenoma to carcinoma in 35% of the cases. Lastly, in half of the malignant polyps studied the main clone in the carcinoma component was already present in the adenoma, although at a low frequency and only detectable by single cell analysis. Conclusion In conclusion our data suggest that CNAs are early events in colorectal carcinogenesis that can determine the evolutionary transition from adenoma to adenocarcinoma.