Spatial Distribution of Private Gene Mutations in Clear Cell Renal Cell Carcinoma

SIMPLE SUMMARY: Tumours consist of multiple groups of similar cells resulting from differing evolutionary trajectories, i.e., subclones. These subclones are prevalent in clear cell renal cell carcinoma (ccRCC). The aim of this study is to determine how similar or dissimilar the subclones in 89 ccRCC tumours are from one another regarding their gene mutations and expression profiles, i.e., the extent of intra-tumour heterogeneity. The implications of these alterations with respect to signalling pathways is also assessed. Deep sequencing allows for the identification of mutations with low-allele frequencies, providing a more comprehensive view of the heterogeneity present in the tumours. With an average of 62% of mutations having been identified in only one of the two biopsies, some of which in turn are found to impact gene expression, the complex makeup of ccRCC tumours is evident, and this can drastically influence treatment outcome. ABSTRACT: Intra-tumour heterogeneity is the molecular hallmark of renal cancer, and the molecular tumour composition determines the treatment outcome of renal cancer patients. In renal cancer tumourigenesis, in general, different tumour clones evolve over time. We analysed intra-tumour heterogeneity and subclonal mutation patterns in 178 tumour samples obtained from 89 clear cell renal cell carcinoma patients. In an initial discovery phase, whole-exome and transcriptome sequencing data from paired tumour biopsies from 16 ccRCC patients were used to design a gene panel for follow-up analysis. In this second phase, 826 selected genes were targeted at deep coverage in an extended cohort of 89 patients for a detailed analysis of tumour heterogeneity. On average, we found 22 mutations per patient. Pairwise comparison of the two biopsies from the same tumour revealed that on average, 62% of the mutations in a patient were detected in one of the two samples. In addition to commonly mutated genes (VHL, PBRM1, SETD2 and BAP1), frequent subclonal mutations with low variant allele frequency (<10%) were observed in TP53 and in mucin coding genes MUC6, MUC16, and MUC3A. Of the 89 ccRCC tumours, 87 (~98%) harboured private mutations, occurring in only one of the paired tumour samples. Clonally exclusive pathway pairs were identified using the WES data set from 16 ccRCC patients. Our findings imply that shared and private mutations significantly contribute to the complexity of differential gene expression and pathway interaction and might explain the clonal evolution of different molecular renal cancer subgroups. Multi-regional sequencing is central for the identification of subclones within ccRCC.