High clonal diversity and spatial genetic admixture in early prostate cancer and surrounding normal tissue

Copy number alterations (CNAs) are pervasive in advanced human cancers, but their prevalence in early-stage, localized tumors and their surrounding normal tissues is poorly characterized. To investigate this phenomenon, here we developed a method for spatially resolved single-cell CNA profiling and applied it to characterize the CNA landscape in 10,007 nuclei extracted from 70 tumor and normal tissue regions (~125 mm3 tissue cubes) from prostatectomies performed in six patients with localized prostate cancer. We identified two distinct groups of cells with abnormal karyotype, one mainly consisting of sparse alterations (‘pseudo-diploid’ cells) and the other characterized by genome-wide karyotypic changes (‘monster’ cells). Pseudo-diploid cells displayed high clonal diversity and formed numerous small sized clones ranging from highly spatially localized to broadly spread clones, whereas monster cells were singular events detected throughout the prostate. We observed a remarkable correlation between the fraction of the genome affected by CNAs and the number of tissue regions in which pseudo-diploid cells were found. Highly localized pseudo-diploid clones were enriched in tumor regions and carried deletions of known or putative tumor suppressors, including APC, CDKN1B, FOXO1, FOXP1, and RB1. Spatially resolved targeted deep sequencing of 523 cancer genes detected non-synonymous mutations in both normal and tumor regions, including mutations in FOXA1, FOXP1, and SPOP genes previously implicated in prostate cancer. Strikingly, in two regions in which targeted deep sequencing detected a point mutation affecting the DNA-binding activity of the FOXA1 transcription factor, we also found a co-deletion of FOXO1 and FOXO3 genes in cells from two different pseudo-diploid clones, implicating combinatorial perturbations of Forkhead transcription factors as an early driver of prostate carcinogenesis. Our study reveals that CNAs and mutations are widespread across normal and tumor regions in the prostate glands of patients with localized prostate cancer and suggests that a subset of alterations—most likely small deletions causing the loss of key tumor suppressors—confer a fitness advantage and channel cells towards tumorigenesis.