Stuart A. McDonald

Mechanisms of field cancerization in the human stomach: the expansion and spread of mutated gastric stem cells.

BACKGROUND & AIMS How mutations are established and spread through the human stomach is unclear because the clonal structure of gastric mucosal units is unknown. Here we investigate, using mitochondrial DNA (mtDNA) mutations as a marker of clonal expansion, the clonality of the gastric unit and show how mutations expand in normal mucosa and gastric mucosa showing intestinal metaplasia. This has important implications in gastric carcinogenesis. METHODS Mutated units were identified by a histochemical method to detect activity of cytochrome c oxidase. Negative units were laser-capture microdissected, and mutations were identified by polymerase chain reaction sequencing. Differentiated epithelial cells were identified by immunohistochemistry for lineage markers. RESULTS We show that mtDNA mutations establish themselves in stem cells within normal human gastric body units, and are passed on to all their differentiated progeny, thereby providing evidence for clonal conversion to a new stem cell-derived unit-monoclonal conversion, encompassing all gastric epithelial lineages. The presence of partially mutated units indicates that more than one stem cell is present in each unit. Mutated units can divide by fission to form patches, with each unit sharing an indentical, mutant mtDNA genotype. Furthermore, we show that intestinal metaplastic crypts are clonal, possess multiple stem cells, and that fission is a mechanism by which intestinal metaplasia spreads. CONCLUSIONS These data show that human gastric body units are clonal, contain multiple multipotential stem cells, and provide definitive evidence for how mutations spread within the human stomach, and show how field cancerization develops.

Intestinal stem cells.

The intestinal tract has a rapid epithelial cell turnover, which continues throughout life. The process is regulated and maintained by a population of stem cells, which give rise to all the intestinal epithelial cell lineages. Studies in both the mouse and the human show that these cells are capable of forming clonal crypt populations. Stem cells remain hard to identify, however it is thought that they reside in a ‘niche’ towards the base of the crypt and their activity is regulated by the paracrine secretion of growth factors and cytokines from surrounding mesenchymal cells. Stem cell division is usually asymmetric with the formation of an identical daughter stem cell and committed progenitor cells. Progenitor cells retain the ability to divide until they terminally differentiate. Occasional symmetric division produces either 2 daughter cells with stem cell loss, or 2 stem cells and eventual clone dominance. This stochastic extinction of stem cell lines with eventual dominance of one cell line is called ‘niche succession’. The discovery of plasticity, the ability of stem cells to engraft into, and in some cases replace the function of damaged host tissues has generated a large amount of scientific and clinical interest: however the concept remains controversial and is still a subject of hot debate. Studies are beginning to identify the complex molecular, genetic and cellular pathways underlying stem cell function such as Wnt signalling, bone morphogenetic protein (BMP) and Notch/Delta pathways. The derangement of these pathways within stem cells plays an integral part in the development of malignancy within the intestinal tract.

Detection of Intestinal Metaplasia in Barrett's Esophagus: An Observational Comparator Study Suggests the Need for a Minimum of Eight Biopsies

OBJECTIVES:Intestinal metaplasia (IM) and dysplasia in Barretts esophagus are recognized surrogates for esophageal adenocarcinoma risk. While few would argue with the “hunt for dysplasia,” there is a divide regarding the usefulness of the histological confirmation of intestinal metaplasia in endoscopically apparent long segment Barretts esophagus. We aimed to assess the frequency of intestinal metaplasia in 125 consecutive patients with columnar-lined esophagus and to determine the optimal biopsy protocol to detect intestinal metaplasia.METHODS:Two-hundred ninety-six endoscopies were performed over a 4-yr period in Barretts esophagus segments of mean length 4 cm (range 1–11 cm) at a single center and the resulting biopsies were analyzed retrospectively. Biopsies were all processed with routine hematoxylin and eosin (H&E) staining, and a subset (N = 92) was subject to alcian blue/periodic-acid Schiff staining.RESULTS:Using H&E staining, we found that the optimum number of biopsies to diagnose intestinal metaplasia was 8 per endoscopy, mean 67.9% endoscopies having intestinal metaplasia. In contrast, if only four were taken the yield was 34.7% with intestinal metaplasia. Unless more than 16 biopsies were taken (100% yield of intestinal metaplasia), no additional significant detection was achieved. Using additional alcian blue/periodic-acid Schiff staining only had a marginal benefit, with 5.4% of new cases of intestinal metaplasia being identified. There is a proximal cephalo-caudal gradient of intestinal metaplasia, especially with increased chronological age, but doing repeat endoscopies on patients did not increase the detection of intestinal metaplasia.CONCLUSIONS:The data suggest that at least 8 random biopsies is the minimum to be taken and analyzed with conventional H&E staining to diagnose benign intestinal metaplasia. Taking more biopsies did not statistically increase the diagnosis of intestinal metaplasia except when greater than 16 were taken when 100% yield was obtained.

Individual crypt genetic heterogeneity and the origin of metaplastic glandular epithelium in human Barrett's oesophagus

Objectives: Current models of clonal expansion in human Barrett’s oesophagus are based upon heterogenous, flow-purified biopsy analysis taken at multiple segment levels. Detection of identical mutation fingerprints from these biopsy samples led to the proposal that a mutated clone with a selective advantage can clonally expand to fill an entire Barrett’s segment at the expense of competing clones (selective sweep to fixation model). We aimed to assess clonality at a much higher resolution by microdissecting and genetically analysing individual crypts. The histogenesis of Barrett’s metaplasia and neo-squamous islands has never been demonstrated. We investigated the oesophageal gland squamous ducts as the source of both epithelial sub-types. Methods: Individual crypts across Barrett’s biopsy and oesophagectomy blocks were dissected. Determination of tumour suppressor gene loss of heterozygosity patterns, p16 and p53 point mutations were carried out on a crypt-by-crypt basis. Cases of contiguous neo-squamous islands and columnar metaplasia with oesophageal squamous ducts were identified. Tissues were isolated by laser capture microdissection and genetically analysed. Results: Individual crypt dissection revealed mutation patterns that were masked in whole biopsy analysis. Dissection across oesophagectomy specimens demonstrated marked clonal heterogeneity, with multiple independent clones present. We identified a p16 point mutation arising in the squamous epithelium of the oesophageal gland duct, which was also present in a contiguous metaplastic crypt, whereas neo-squamous islands arising from squamous ducts were wild-type with respect to surrounding Barrett’s dysplasia. Conclusions: By studying clonality at the crypt level we demonstrate that Barrett’s heterogeneity arises from multiple independent clones, in contrast to the selective sweep to fixation model of clonal expansion previously described. We suggest that the squamous gland ducts situated throughout the oesophagus are the source of a progenitor cell that may be susceptible to gene mutation resulting in conversion to Barrett’s metaplastic epithelium. Additionally, these data suggest that wild-type ducts may be the source of neo-squamous islands.

Clonality, Founder Mutations, and Field Cancerization in Human Ulcerative Colitis–Associated Neoplasia

BACKGROUND & AIMS The clonality of colitis-associated neoplasia has not been fully determined. One previous report showed polyclonal origins with subsequent monoclonal outgrowth. We aimed to assess the clonality and mutation burden of individual crypts in colitis-associated neoplasias to try to identify gatekeeping founder mutations, and explore the clonality of synchronous lesions to look for field effects. METHODS Individual crypts (range, 8-21 crypts) were microdissected from across 17 lesions from 10 patients. Individual crypt adenomatous polyposis coli (APC), p53, K-RAS, and 17p loss of heterozygosity mutation burden was established using polymerase chain reaction and sequencing analysis. Serial sections underwent immunostaining for p53, beta-catenin, and image cytometry to detect aneuploidy. RESULTS In most lesions an oncogenic mutation could be identified in all crypts across the lesion showing monoclonality. This founder mutation was a p53 lesion in the majority of neoplasms but 4 tumors had an initiating K-RAS mutation. Some nondysplastic crypts surrounding areas of dysplasia were found to contain clonal p53 mutations and in one case 3 clonal tumors arose from a patch of nondysplastic crypts containing a K-RAS mutation. CONCLUSIONS This study used mutation burden analysis of individual crypts across colitis-associated neoplasms to show lesion monoclonality. This study confirmed p53 mutation as initiating mutation in the majority of lesions, but also identified K-RAS activation as an alternative gatekeeping mutation. Local and segmental field cancerization was found by showing pro-oncogenic mutations in nondysplastic crypts surrounding neoplasms, although field changes are unlikely to involve the entire colon because widely separated tumors were genetically distinct.

Locating the stem cell niche and tracing hepatocyte lineages in human liver.

We have used immunohistochemical and histochemical techniques to identify patches of hepatocytes deficient in the enzyme cytochrome c oxidase, a component of the electron transport chain and encoded by mitochondrial DNA (mtDNA). These patches invariably abutted the portal tracts and expanded laterally as they spread toward the hepatic veins. Here we investigate, using mtDNA mutations as a marker of clonal expansion, the clonality of these patches. Negative hepatocytes were laser‐capture microdissected and mutations identified by polymerase chain reaction sequencing of the entire mtDNA genome. Patches of cytochrome c oxidase–deficient hepatocytes were clonal, suggesting an origin from a long‐lived cell, presumably a stem cell. Immunohistochemical analysis of function and proliferation suggested that these mutations in cytochrome c oxidase‐deficient hepatocytes were nonpathogenic. Conclusion: these data show, for the first time, that clonal proliferative units exist in the human liver, an origin from a periportal niche is most likely, and that the trajectory of the units is compatible with a migration of cells from the periportal regions to the hepatic veins. (HEPATOLOGY 2009.)

Clonality Assessment and Clonal Ordering of Individual Neoplastic Crypts Shows Polyclonality of Colorectal Adenomas

BACKGROUND & AIMS According to the somatic mutation theory, monoclonal colorectal lesions arise from sequential mutations in the progeny of a single stem cell. However, studies in a sex chromosome mixoploid mosaic (XO/XY) patient indicated that colorectal adenomas were polyclonal. We assessed adenoma clonality on an individual crypt basis and completed a genetic dependency analysis in carcinomas-in-adenomas to assess mutation order and timing. METHODS Polyp samples were analyzed from the XO/XY individual, patients with familial adenomatous polyposis and attenuated familial adenomatous polyposis, patients with small sporadic adenomas, and patients with sporadic carcinoma-in-adenomas. Clonality was analyzed using X/Y chromosome fluorescence in situ hybridization, analysis of 5q loss of heterozygosity in XO/XY tissue, and sequencing of adenomatous polyposis coli. Individual crypts and different phenotypic areas of carcinoma-in-adenoma lesions were analyzed for mutations in adenomatous polyposis coli, p53, and K-RAS; loss of heterozygosity at 5q, 17p, and 18q; and aneuploidy. Phylogenetic trees were constructed. RESULTS All familial adenomatous polyposis-associated adenomas and some sporadic lesions had polyclonal genetic defects. Some independent clones appeared to be maintained in advanced adenomas. No clear obligate order of genetic events was established. Top-down growth of dysplastic tissue into neighboring crypts was a possible mechanism of clonal competition. CONCLUSIONS Human colorectal microadenomas are polyclonal and may arise from a combination of host genetic features, mucosal exposures, and active crypt interactions. Analyses of tumor phylogenies show that most lesions undergo intermittent genetic homogenization, but heterotypic mutation patterns indicate that independent clonal evolution can occur throughout adenoma development. Based on observations of clonal ordering the requirement and timing of genetic events during neoplastic progression may be more variable than previously thought.

Quantification of Crypt and Stem Cell Evolution in the Normal and Neoplastic Human Colon

Summary Human intestinal stem cell and crypt dynamics remain poorly characterized because transgenic lineage-tracing methods are impractical in humans. Here, we have circumvented this problem by quantitatively using somatic mtDNA mutations to trace clonal lineages. By analyzing clonal imprints on the walls of colonic crypts, we show that human intestinal stem cells conform to one-dimensional neutral drift dynamics with a “functional” stem cell number of five to six in both normal patients and individuals with familial adenomatous polyposis (germline APC−/+). Furthermore, we show that, in adenomatous crypts (APC−/−), there is a proportionate increase in both functional stem cell number and the loss/replacement rate. Finally, by analyzing fields of mtDNA mutant crypts, we show that a normal colon crypt divides around once every 30–40 years, and the division rate is increased in adenomas by at least an order of magnitude. These data provide in vivo quantification of human intestinal stem cell and crypt dynamics.

Mechanisms of Disease: from stem cells to colorectal cancer

Over the past decade, the advances in our understanding of stem cell biology and the role of stem cells in diseases, such as colorectal cancer, have been remarkable. In particular, discoveries related to the control of stem cell proliferation and how dysregulation of proliferation leads to oncogenesis have been foremost. For intestinal stem cells, the WNT family of growth factors, and events such as the regulation of the nuclear localization of β-catenin, seem to be central to normal homeostasis, and mutations in the components of these pathways seem to lead to the development of colorectal cancer. A paradigm of abnormal stem cell biology is illustrated by patients with familial adenomatous polyposis, who have mutations in the adenomatous polyposis coli gene. The wild-type protein encoded by this gene is important for the prevention of mass β-catenin accumulation in the nucleus and the subsequent overtranscription of cell cycle proteins. This review discusses the basic mechanisms behind stem cell regulation in the gut and follows their role in the natural history of tumor progression.

Characterization of the mucosal cell‐mediated immune response in IL‐2 knockout mice before and after the onset of colitis

One of the major advances in the understanding of inflammatory bowel disease has been the observation that mice with immunoregulatory defects, such as interleukin‐2 knockout (IL‐2 –/–) mice, develop spontaneous gut inflammation. Here we have characterized the immune response in the ileum, caecum and colon of these mice before and after the onset of colitis by examining the cellular infiltrate, the cytokines produced by these cells and the mucosal vascular addressin MAdCAM‐1. IL‐2 –/– mice developed colitis after 35 days of age and before this the mice were apparently healthy. IL‐2 –/– mice aged over 35 days with colitis had large numbers of CD4+, CD8+, αβ T‐cell receptor (TCR)+ and γδ TCR+ T cells, macrophages, dendritic cells and MAdCAM‐1+ endothelial cells in the caecum and colon. This was associated with an increase in the number of interferon‐γ (IFN‐γ), IL‐1 and tumour necrosis factor‐α (TNF‐α) transcripts and a decrease in IL‐4 and IL‐10 transcripts. Treatment of IL‐2 –/– mice with cyclosporin A significantly delayed mortality. Interestingly, IL‐2 –/– mice under 35 days, although healthy, did show some subtle immunological signs of preclinical disease. There was a significant increase in the number of macrophages and dendritic cells in the colonic lamina propria and increased mRNA for IL‐1 and TNF‐α. There were also increased numbers of MAdCAM‐1+ endothelial cells, but IFN‐γ transcripts were not elevated. These results suggest that T‐cell‐mediated colitis in IL‐2 –/– mice may be secondary to an initial non‐specific inflammation.