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...

Diseases of esophageal epithelium (EE) such as reflux esophagitis and cancer are rising in incidence. Despite this, the cellular behaviors underlying EE homeostasis and repair remain controversial. Here we show that in mice, EE is maintained by a single population of cells that divide stochastically to generate proliferating and differentiating daughters with equal probability. In response to challenge with all-trans Retinoic Acid (atRA) the balance of daughter cell fate is unaltered but the rate of cell division increases. However, following wounding, cells reversibly switch to producing an excess of proliferating daughters until the wound has closed. Such fate switching enables a single progenitor population to both maintain and repair tissue without the need for a "reserve" slow-cycling stem cell pool.Murine EE consists of layers of keratinocytes. These tissue lacks structures such as crypts or glands which form stem cell niches in other epithelia . Proliferation is confined to cells in the basal layer (6). On commitment to terminal differentiation, basal cells exit the cell cycle and subsequently migrate to the tissue surface from which they are shed. Early studies suggested all proliferating cells were functionally equivalent, but recent reports propose that a discrete population of slow-cycling stem cells is responsible for both maintenance and wound healing (7)(8)(9)(10)(11). This controversy and the importance of EE in disease motivated us to resolve the proliferative cell behavior in homeostatic EE and in tissue challenged by systemic treatment with the vitamin A metabolite all-trans Retinoic Acid (atRA) or acute local wounding (12-13).To investigate cell division rates in EE we used a transgenic label retaining cell (LRC) assay ( Fig. 1C) (1, 14-15). Doxycycline (DOX) induction of Histone-2B EGFP fusion protein (HGFP) expression in Rosa26 M2rtTA /TetO-HGFP mice resulted in nuclear fluorescent labeling throughout the EE (Fig. 1D and fig. S1A). When DOX is withdrawn, HGFP is diluted by cell division, leaving 0.4% basal layer cells (561 out of 140000) retaining label after a 4 week chase (Figs. 1E and S1B). 3D imaging showed these label retaining cells * To whom correspondence should be addressed. phj20@cam.ac.uk. 5 These authors contributed equally to this work Supplementary Materials: Materials and Methods Figures S1-S13 However, 99.9% (2457 out of 2459) of LRC were positive for the pan leukocyte marker CD45 (Fig. 1E inset), comprising of a mixture of Langerhan's cells and lymphocytes (Figs. S1E and F). These findings lead to the surprising conclusion that, unlike tissues such as the epidermis, there are no slow-cycling or quiescent epithelial stem cells in EE (1, 17). Indeed, HGFP dilution in basal cells was strikingly homogeneous, suggesting that all cells divide at a similar rate of approximately twice per week (Fig. S1G).Although epithelial cells have the same rate of division, they may still differ in their ability to generate cycling and differentiated progeny. We therefore used inducible ...