Volume 134, Issue 2, Pages 500-510 (February 2008) Mechanisms of Field Cancerization in the Human Stomach: The Expansion and Spread of Mutated Gastric Stem Cells  Stuart A.C. McDonald, Laura C. Greaves, Lydia Gutierrez–Gonzalez, Manuel Rodriguez–Justo, Maesha Deheragoda, Simon J. Leedham, Robert W. Taylor, Chung Yin Lee, Sean L. Preston, Matthew Lovell, Toby Hunt, George Elia, Dahmane Oukrif, Rebecca Harrison, Marco R. Novelli, Ian Mitchell, David L. Stoker, Douglass M. Turnbull, Janusz A.Z. Jankowski, Nicholas A. Wright  Gastroenterology  Volume 134, Issue 2, Pages 500-510 (February 2008) DOI: 10.1053/j.gastro.2007.11.035 Copyright © 2008 AGA Institute Terms and Conditions

Figure 1 Cytochrome c oxidase/SDH enzyme histochemistry on human stomach sections. Partial cytochrome c oxidase–deficient gastric units are observed. (A) A single deficient cell is seen within an otherwise positive unit in transverse section. Because there are both mutated and nonmutated cells in the same unit, we conclude that there are at least 2 stem cells present. Inspection of E shows that the unit marked § is partially mutated and probably in the process of conversion. (A) A unit cut in cross-section, and thus the mutated cell shown is the progeny of a mutated stem cell in the neck of the unit. (B) A partially mutated gastric unit showing a restricted phenotype (arrows). The unit is entirely negative from the neck down (top arrow), but the foveolar cells are positive. Entirely cytochrome c oxidase–deficient gastric units also were observed, showing the process of monoclonal conversion. (C) A cytochrome c oxidase–deficient unit in cross-section around the gland region. (D) A gastric unit in which all the gastric epithelial lineages are mutated (black arrow) apart from the parietal cells, which are wild-type, and stain brown (white arrow). (E) Two gastric units, one is partially cytochrome c oxidase deficient (§) and the other is entirely deficient (*). Although the staining of the foveola is less intense than the rest of the unit because of fewer mitochondria contained within foveolar cells, the foveolus can be seen clearly in both units, and therefore are distinct units. Whole-mount enzyme histochemistry was performed on EDTA-extracted gastric units showing the presence of entirely (F) positive and (G) deficient units. This is strong evidence that human gastric units are clonal and contain multiple stem cells. Gastroenterology 2008 134, 500-510DOI: (10.1053/j.gastro.2007.11.035) Copyright © 2008 AGA Institute Terms and Conditions

Figure 2 Normal human gastric units are clonal. (A) A cytochrome c oxidase–deficient gastric unit. (B) Negative cells have been captured from the foveolus (1), around the neck region (2) and base (3) of a negative unit, and also from the base (4) of a neighboring positive unit. (C) Sequencing revealed a T>C transition at position 8181, resulting in an I>T amino acid change in complex IV (cytochrome c oxidase genes). Gastroenterology 2008 134, 500-510DOI: (10.1053/j.gastro.2007.11.035) Copyright © 2008 AGA Institute Terms and Conditions

Figure 3 Gland fission is a mechanism of mutation spread in the human stomach. (A) An entirely cytochrome c oxidase–deficient patch (on a laser-capture membrane slide). (B) Single cells from each gastric unit (a deficient unit indicated by a black arrow, a cytochrome c oxidase–normal gastric unit indicated by a white arrow) within the patch and their cytochrome c oxidase–positive neighboring units were laser capture–microdissected and the entire mitochondrial genome was sequenced. (C) All the cytochrome c oxidase–deficient cells contained the same 2593G>A transition mutation (black arrow), which was not detected in any of the neighboring cytochrome c oxidase–positive cells (D, white arrow). Importantly, this was the only nucleotide position at which mtDNA sequences from cytochrome c oxidase–deficient cells differed from those obtained in cytochrome c oxidase–positive cells. Gastroenterology 2008 134, 500-510DOI: (10.1053/j.gastro.2007.11.035) Copyright © 2008 AGA Institute Terms and Conditions

Figure 4 All differentiated epithelial cell lineages in a gastric unit have a common ancestor. (A) Immunohistochemistry of a cytochrome c oxidase subunit 1–deficient patch. The brown foveolus on the left-hand side of the mutated patch belongs to a wild-type foveolus from a gastric unit out of the plane of section: looking to the right of the figure, the foveolus here is negative. The units contained the following: (B) H+K+ATPase+ve parietal cells, (C) chromogranin A+ve neuroendocrine cells, (D) a small number of TFF-1+ve foveolar cells, and (E) pepsinogen I+ve chief (and neck) cells. Note that some of the mucous neck cells also are stained, which is consistent with the Karam and Leblond concept of the chief cell being derived from the mucous neck cell.26,27 (F) TFF-2+ve mucus neck cells. (G and H) Anti-mouse and anti-sheep immunoglobulin (Ig)G–matched isotype controls. A small simple cyst is present. However, this is a common occurrence within the stomach cardia, and no other cysts in this tissue block were cytochrome c oxidase deficient. Gastroenterology 2008 134, 500-510DOI: (10.1053/j.gastro.2007.11.035) Copyright © 2008 AGA Institute Terms and Conditions

Figure 5 Cytochrome c oxidase/SDH histochemistry on sections of human IM of the stomach. (A) Entirely cytochrome c oxidase–deficient crypt adjacent to a positive one. (B) Partially cytochrome c oxidase–deficient crypt (*some cells toward the base are positive and some are positive in the high-power insert). This is strong evidence that intestinal metaplastic crypts are clonal, contain multiple stem cells, and can undergo monoclonal conversion. All the major differentiated intestinal epithelial cell lineages are present in cytochrome c oxidase–deficient metaplastic crypts. By immunohistochemistry, (C) cytochrome c oxidase, (D) MUC2+ve goblet cells, (E) chromogranin A+ve neuroendocrine cells, (F) IgG-matched isotype control, (G) CD10+ve enterocytes, and (H) Alcian blue/diastase stain revealing goblet cells. Gastroenterology 2008 134, 500-510DOI: (10.1053/j.gastro.2007.11.035) Copyright © 2008 AGA Institute Terms and Conditions

Figure 6 Patches of cytochrome c oxidase–deficient metaplastic crypts are clonal and expand by fission. (A) A patch of deficient crypts (blue) pre–laser capture. (B) Post–laser capture (1–3, blue; 4 and 5, brown). (C) Sequencing revealed a T>C transition (at position 8503 in the mtATP8 gene) in the cytochrome c oxidase–deficient cells (1 and 3 shown here), but not in the cytochrome c oxidase–positive cells (5 shown here). Gastroenterology 2008 134, 500-510DOI: (10.1053/j.gastro.2007.11.035) Copyright © 2008 AGA Institute Terms and Conditions

Figure 7 Schematics of the central hypotheses of this article. (A) Monoclonal conversion: the process begins with a mutation resulting in cytochrome c oxidase deficiency establishing itself in a stem cell (*within the isthmus/neck region of the gastric unit). Stochastically this stem cell takes over the entire stem cell zone, the progeny of which migrate up the foveolus and down the gland (left to right) until the gastric unit becomes entirely mutated. It should be noted that a partially mutated unit may convert back to a wild-type phenotype because we do not believe these mutations confer a selective advantage. (B) Gland fission: patches develop by budding from the isthmus/neck region into a new gland (left to right) that eventually develops its own foveolus and hence a new gastric unit is formed. The molecular mechanisms of how fission is initiated are unknown. Gastroenterology 2008 134, 500-510DOI: (10.1053/j.gastro.2007.11.035) Copyright © 2008 AGA Institute Terms and Conditions