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 ...

SummarySingle stem cells, including those in human epidermis, have a remarkable ability to reconstitute tissues in vitro, but the cellular mechanisms that enable this are ill defined. We used live imaging to track the outcome of thousands of divisions in clonal cultures of primary human epidermal keratinocytes. Two modes of proliferation were seen. In ‘balanced’ mode, similar proportions of proliferating and differentiating cells were generated, achieving the ‘population asymmetry’ that sustains epidermal homeostasis in vivo. In ‘expanding’ mode, an excess of cycling cells was produced, generating large expanding colonies. Cells in expanding mode switched their behaviour to balanced mode once local confluence was attained. However when a confluent area is wounded in a scratch assay, cells near the scratch switch back to expanding mode until the defect is closed. We conclude that the ability of a single epidermal stem cell to reconstitute an epithelium is explained by two interconvertible modes of proliferation regulated by confluence.

Skin cancer risk varies substantially across the body, yet how this relates to the mutations found in normal skin is unknown. Here we mapped mutant clones in skin from high and low risk sites. The density of mutations varied by location. The prevalence of NOTCH1 and FAT1 mutations in forearm, trunk and leg skin was similar to that in keratinocyte cancers. Most mutations were caused by ultraviolet (UV) light, but mutational signature analysis suggested differences in DNA repair processes between sites. 11 mutant genes were under positive selection, with TP53 preferentially selected in the head and FAT1 in the leg. Fine scale mapping revealed 10% of clones had copy number alterations. Analysis of hair follicles showed mutations in the upper follicle resembled adjacent skin, but the lower follicle was sparsely mutated. Normal skin is dense patchwork of mutant clones arising from competitive selection that varies by location. Statement of significance Mapping mutant clones across the body reveals normal skin is a dense patchwork of mutant cells. The variation in cancer risk between sites substantially exceeds that in mutant clone density. More generally, mutant genes cannot be assigned as cancer drivers until their prevalence in normal tissue is known.

A neglected femoral neck fracture is one where there has been a delay of more than 30 days to seek medical help from the time of the original injury. Among the spectrum of femoral neck fractures, the neglected fracture in a young adult (age <60 years) is one of the most challenging to treat if femoral head salvage is attempted.The main complication is avascular necrosis (AVN) of the femoral head with most reported incidences being <15% (range 0% to 67%), which is similar to the complication rate with non-neglected femoral neck fractures.This review consolidates our current knowledge about the problem, discusses the various treatment options and compares the published long-term results.There are no clear guidelines for management of neglected femoral neck fractures, although multiple methods have been used with varying success. Bone grafting or internal fixation in isolation does not provide a satisfactory outcome. Osteotomy has given better outcomes (AVN 6% to 17%, non-union 0% to 15%), but mechanical changes at the femoral neck may lead to a persistent painful hip. Bone grafting with internal fixation has emerged as a reliable method with good long-term functional outcomes.

Neglected femoral fractures in young adults are a challenge to the orthopaedic surgeon, requiring prolonged treatment and with attendant risks of nonunion. We postulated treatment in this group by accurate reduction, two cannulated screws, and whole free fibular autograft would allow early mobilization and provide good bony union. Thirty-two patients aged 18 to 50 years were treated at our center in this manner. They presented to our center 3 to 6 months after injury, and had Garden's Grade III/IV fractures with varying degrees of neck resorption, but no avascular necrosis. No plaster was applied, and early return to function was encouraged. Bony union was achieved in 29 (90.6%) patients at a mean of 19.2 weeks (range, 16-24 weeks). All patients with union had good function at long-term followup at an average of 6.1 years postoperatively (range, 2-12 years), and the average Harris hip score was 87.1 points (range, 74.5-94 points). Our procedure allows early return of function in young, active patients disabled by old femoral neck fractures compounded by lack of early treatment.

Ann RC oll Surg Engl 2008; 90:6 01-605 601The explosion of internet usage by patients and the amount of healthcare information publicly available has led to new challenges for practising physicians. 1,2 The information available from at hird party has important implications for the doctor-patient relationship and can be used to enhance the healthcare experience. 2,3 Freedom of information on the internet dictates that anyone can publish information. Therefore, there is ar isk that such information, through ignorance or bias, may be incorrect or misleading. [4][5][6] Information of dubious quality or that is commercially motivated can be misleading, or even potentially lethal. 7,8 Many tools are available to assess healthcare information on the internet and, although there is consensus on key criteria, they are still being developed. 9-11 The LIDA Instrument is avalidated method of evaluating healthcare websites based on three important areas -a ccessibility,u sability and reliability. 12 Search engines are the most commonly used method to find information on the world wide web. Information exists about the usage characteristics of search engines in the UK. Ratings such as the Neilson'sN et rating 13 provide data for the most widely used search engines, and patterns therein. The five most visited search engines (Google, Yahoo, MSN, AOL and AskJeeves) field >9 0% of all search questions according to these surveys. Although searches often reveal thousands of links, at ypical user is likely to visit the most The aim of this studyw as to assess the quality of information available on the world wide web to parents of children undergoing tonsillectomy. HEAD &N ECK

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