How somatic mutations accumulate in normal cells is central to understanding cancer development, but is poorly understood. We performed ultra-deep sequencing of 74 cancer genes in small (0.8-4.7mm 2 ) biopsies of normal skin. Across 234 biopsies of sun-exposed eyelid epidermis from four individuals, the burden of somatic mutations averaged 2-6 mutations/megabase/cell, similar to many cancers, and exhibited characteristic signatures of ultraviolet light exposure. Remarkably, multiple cancer genes are under strong positive selection even in physiologically normal skin, including most of the key drivers of cutaneous squamous cell carcinomas. Positively selected 'driver' mutations were found in 18-32% of normal skin cells at a density of ~140/cm 2 . We observed variability in the driver landscape among individuals and variability in sizes of clonal expansions across genes. Thus, aged, sun-exposed skin is a patchwork of thousands of evolving clones, with over a quarter of cells carrying cancer-causing mutations while maintaining the physiological functions of epidermis.The standard narrative of tumor evolution depicts accumulation of driver mutations in cancer genes, causing waves of expansion of progressively more disordered clones (1, 2). Central to this model is the presumption that randomly distributed somatic mutations must accumulate in normal cells before transformation (3), but directly observing them has proved challenging due to the polyclonal composition of normal tissue. Retrospective reconstructions of clonal evolution from sequencing of tumors give only partial insights, leaving us with fundamental gaps in our understanding of the earliest stages of cancer development. Critical, but largely unanswered, questions include the burden of somatic mutations in normal cells, which mutational processes are operative in normal tissues, the extent of positive selection among competing clones within a organ, and the patterns of * Correspondence to: phj20@mrc-cu.cam.ac.uk; pc8@sanger.ac.uk. Europe PMC Funders Group Europe PMC Funders Author ManuscriptsEurope PMC Funders Author Manuscripts clonal expansion induced by the very first driver mutations (4, 5). These questions have been partially addressed in blood cells, where somatic mutations, including some driver mutations, have been found to accumulate at a low rate with increasing age (6-10).To study the burden, mutational processes and clonal architecture of somatic mutations in normal non-hematological tissue, we focused on sun-exposed skin. Previous studies have reported the existence of clonal patches of skin cells carrying TP53 mutations (11)(12)(13)(14)(15). Motivated by this, we designed a sequencing strategy capable of detecting such clones by performing ultra-deep sequencing of small biopsies and adapting algorithms to detect mutations in a small fraction of cells. We used eyelid epidermis because of its relatively high levels of sun exposure and being one of the few body sites to have normal skin excised (blepharoplasty). This procedure is perfo...

During the course of a lifetime somatic cells acquire mutations. Different mutational processes may contribute to the mutations accumulated in a cell, with each imprinting a mutational signature on the cell's genome. Some processes generate mutations throughout life at a constant rate in all individuals and the number of mutations in a cell attributable to these processes will be proportional to the chronological age of the person. Using mutations from 10,250 cancer genomes across 36 cancer types, we investigated clock-like mutational processes that have been operating in normal human cells. Two mutational signatures show clock-like properties. Both exhibit different mutation rates in different tissues. However, their mutation rates are not correlated indicating that the underlying processes are subject to different biological influences. For one signature, the rate of cell division may influence its mutation rate. This study provides the first survey of clock-like mutational processes operative in human somatic cells.