Anna M. Nicholson

Barrett's metaplasia glands are clonal, contain multiple stem cells and share a common squamous progenitor

Background Little is known about the stem cell organisation of the normal oesophagus or Barretts metaplastic oesophagus. Using non-pathogenic mitochondrial DNA mutations as clonal markers, the authors reveal the stem cell organisation of the human squamous oesophagus and of Barretts metaplasia and determine the mechanism of clonal expansion of mutations. Methods Mutated cells were identified using enzyme histochemistry to detect activity of cytochrome c oxidase (CCO). CCO-deficient cells were laser-captured and mutations confirmed by PCR sequencing. Cell lineages were identified using immunohistochemistry. Results The normal squamous oesophagus contained CCO-deficient patches varying in size from around 30 μm up to about 1 mm. These patches were clonal as each area within a CCO-deficient patch contained an identical mitochondrial DNA mutation. In Barretts metaplasia partially CCO-deficient glands indicate that glands are maintained by multiple stem cells. Wholly mutated Barretts metaplasia glands containing all the expected differentiated cell lineages were seen, demonstrating multilineage differentiation from a clonal population of Barretts metaplasia stem cells. Patches of clonally mutated Barretts metaplasia glands were observed, indicating glands can divide to form patches. In one patient, both the regenerating squamous epithelium and the underlying glandular tissue shared a clonal mutation, indicating that they are derived from a common progenitor cell. Conclusion In normal oesophageal squamous epithelium, a single stem cell clone can populate large areas of epithelium. Barretts metaplasia glands are clonal units, contain multiple multipotential stem cells and most likely divide by fission. Furthermore, a single cell of origin can give rise to both squamous and glandular epithelium suggesting oesophageal plasticity.

Field Cancerization in the Intestinal Epithelium of Patients With Crohn's Ileocolitis

BACKGROUND & AIMS Tumors that develop in patients with Crohns disease tend be multifocal, so field cancerization (the replacement of normal cells with nondysplastic but tumorigenic clones) might contribute to intestinal carcinogenesis. We investigated patterns of tumor development from pretumor intestinal cell clones. METHODS We performed genetic analyses of multiple areas of intestine from 10 patients with Crohns disease and intestinal neoplasia. Two patients had multifocal neoplasia; longitudinal sections were collected from 3 patients. Individual crypts were microdissected and genotyped; clonal dependency analysis was used to determine the order and timing of mutations that led to tumor development. RESULTS The same mutations in KRAS, CDKN2A(p16), and TP53 that were observed in neoplasias were also present in nontumor, nondysplastic, and dysplastic epithelium. In 2 patients, carcinogenic mutations were detected in nontumor epithelium 4 years before tumors developed. The same mutation (TP53 p.R248W) was detected at multiple sites along the entire length of the colon from 1 patient; it was the apparent founder mutation for synchronous tumors and multiple dysplastic areas. Disruption of TP53, CDKN2A, and KRAS were all seen as possible initial events in tumorigenesis; the sequence of mutations (the tumor development pathway) differed among lesions. CONCLUSIONS Pretumor clones can grow extensively in the intestinal epithelium of patients with Crohns disease. Segmental resections for neoplasia in patients with Crohns disease might therefore leave residual pretumor disease, and dysplasia might be an unreliable biomarker for cancer risk. Characterization of the behavior of pretumor clones might be used to predict the development of intestinal neoplasia.

The stem cell organisation, and the proliferative and gene expression profile of Barrett's epithelium, replicates pyloric-type gastric glands

Objective Barretts oesophagus shows appearances described as ‘intestinal metaplasia’, in structures called ‘crypts’ but do not typically display crypt architecture. Here, we investigate their relationship to gastric glands. Methods Cell proliferation and migration within Barretts glands was assessed by Ki67 and iododeoxyuridine (IdU) labelling. Expression of mucin core proteins (MUC), trefoil family factor (TFF) peptides and LGR5 mRNA was determined by immunohistochemistry or by in situ hybridisation, and clonality was elucidated using mitochondrial DNA (mtDNA) mutations combined with mucin histochemistry. Results Proliferation predominantly occurs in the middle of Barretts glands, diminishing towards the surface and the base: IdU dynamics demonstrate bidirectional migration, similar to gastric glands. Distribution of MUC5AC, TFF1, MUC6 and TFF2 in Barretts mirrors pyloric glands and is preserved in Barretts dysplasia. MUC2-positive goblet cells are localised above the neck in Barretts glands, and TFF3 is concentrated in the same region. LGR5 mRNA is detected in the middle of Barretts glands suggesting a stem cell niche in this locale, similar to that in the gastric pylorus, and distinct from gastric intestinal metaplasia. Gastric and intestinal cell lineages within Barretts glands are clonal, indicating derivation from a single stem cell. Conclusions Barretts shows the proliferative and stem cell architecture, and pattern of gene expression of pyloric gastric glands, maintained by stem cells showing gastric and intestinal differentiation: neutral drift may suggest that intestinal differentiation advances with time, a concept critical for the understanding of the origin and development of Barretts oesophagus.

Acid Reflux and Oesophageal Cancer

Barretts metaplasia is one of the commonest premalignant lesions in the western world following colorectal adenomas. One in 50 of the adult population develops Barretts as a consequence of chronic gastro-oesophageal reflux. The mucosal inflammation seen within patients with gastro-oesophageal reflux seems likely to drive the growth of the metaplastic mucosa and also help direct further oncological change, yet the molecular events that characterize the pathway from inflammation to metaplasia to dysplasia and adenocarcinoma are poorly understood. There is hope that understanding the role of oesophageal inflammation will provide important insight into the development of Barretts metaplasia and oesophageal cancer. This chapter will discuss the inflammation seen within context of Barretts oesophagus and also clinical trials which hope to address this common premalignant disease. There are several ongoing clinical trials which are aiming to provide data using anti-inflammatory therapies to tackle this important premalignant condition. There is new data presented which suggests that data from the aspirin esomeprazole chemoprevention trial (AspECT) may hold the clue to disease treatment and that the cytokine TNF-α seems to be a key signalling molecule in the metaplasia-dysplasia-carcinoma sequence. Specifically it appears that both epigenetic and inherited genetics cooperate to modulate the prognosis.

Cytoplasmic β‐catenin accumulation is a good prognostic marker in upper and lower gastrointestinal adenocarcinomas

Norwood M G A, Bailey N, Nanji M, Gillies R S, Nicholson A, Ubhi S, Darnton J J, Steyn R S, Womack C, Hughes A, Hemingway D, Harrison R, Waters R & Jankowski J A
(2010) Histopathology 57, 101–111
Cytoplasmic β‐catenin accumulation is a good prognostic marker in upper and lower gastrointestinal adenocarcinomas

Editorial: One Small Step for Metaplasia, but One Giant Leap for Biomarkers Is Needed

There are 5 to 6 levels of biomarker validation. Those for Barretts esophagus are currently at level 3, despite small prospective studies. What is ideally required is a very large prospective assessment of biopsies in large cohorts, such as the ASPirin Esomeprazole Chemoprevention Trial (AspECT) and Barretts Oesophagus Surveillance Study (BOSS) trials, so that unbiased and random selection of cases can be subjected to rigorous pathology and biomarker assessment (level 4). Only then can the predictive power of the data be exploited in a randomized intervention trial (level 5) whereby a series of biomarkers would trigger therapy. The real trouble is that this spot is currently occupied, satisfactorily according to some researchers, by conventional histological identification of high-grade dysplasia (HGD) as used in a recent randomized study of ablation in Barretts esophagus (BE).

Altered stem cell dynamics in human colon adenoma crypts allow rapid expansion and fixation of mutations during clonal expansion

Introduction Methylation patterns at CpG islands within non-expressed genes are surrogate markers of cell ancestry and dynamics in normal human colon crypts, and have been used to provide evidence that crypts contain multiple stem cells within a niche.1 Over time, a stem cell and its progeny will come to occupy the niche- niche succession , and subsequently the crypt- monoclonal conversion . Crypts divide by crypt fission , whereby a parent crypt produces two clonally related daughter crypts; this is the predominant method by which crypts expand in early tumorigenesis.2 By analysing methylation patterns of normal human colon crypts, known to be related by their sharing clonal point mutations in mitochondrial DNA (mtDNA), we found that the rate of niche succession is slow (years) and crypt fission is rare.3 There is a need to evaluate these dynamics in adenomas to understand how quickly they grow; we hypothesise that both stem cell niche succession and crypt fission occur at a faster rate in adenoma tissue enabling accelerated clonal expansion. Methods Fresh frozen human adenomas were obtained and clonal patches of adenomatous crypts identified by combining enzyme histochemistry and laser capture microdissection with the use of PCR sequencing to demonstrate shared clonal point mutations in mtDNA. Using DNA from the same crypt, methylation patterns at CpG islands of three non-expressed genes were determined using clonal bisulphite sequencing. Results In contrast to normal human colon, multiple, large clonal patches of crypts that shared common point mutations in their mtDNA were identified within human adenomas; these recently related adenomatous crypts had very similar, conserved methylation patterns compared to unrelated crypts (p=8.08×10 −12 ). Conclusion This study demonstrates that increased rates of stem cell niche succession, monoclonal conversion and crypt fission enable accelerated clonal expansion of mutated crypts within human adenomas, suggesting that these up-regulated processes allow rapid fixation and spread of successive oncogenic mutations during early tumorigenesis; further data collection will allow the time course of these events to be estimated. Combining the techniques used here with clonal oncogenic changes may reveal the selective growth advantage of specific mutations within adenomas.

947 Analysis of the Clonality of Barrett's Esophagus Glands Reveals They are Clonal Units and Establishes a Common Stem Cell for Glandular and Squamous Epithelium

Introduction Barrett9s oesophagus (BE) describes the change from the normal keratinised squamous epithelium of the oesophagus into an intestinal columner phenotype. BE is the only known precursor of oesophageal adenocarcinoma. Little is known about the origin and stem cell architecture of BE glands, or about the mechanism of gland spread. Here we have used non-pathologic mitochondrial DNA (mtDNA) mutations as a marker of clonal expansion to investigate the origin and spread of clones within BE. Methods Enzyme histochemistry for cytochrome-c oxidase (CCO) and succinate dehydrogenase (SDH) was performed on frozen sections of BE. Laser-capture microdissected cells were taken from glandular and squamous epithelium. Mutations were indentified using a nested PCR protocol and sequencing of the entire mtDNA. Results CCO deficient glands, were seen in sections of a gastric type BE (n=2/2), but not within intestinal metaplasia (n=3/3). Where CCO deficient glands were seen all cells contained the same mutation, suggesting a common stem cell origin. Mixed glands containing CCO positive and negative lineages were observed suggesting multiple stem cells may be present in the one gland. Patches of adjacent glands varying between 2-7 glands containing the same mutation were seen, indicating that BE glands spread by fission. In one case, cells from CCO deficient squamous tissue shared the same mtDNA mutation as cells from underlying CCO deficient glandular epithelium, suggesting the two tissue compartments are derived from a common progenitor cell Conclusion mtDNA mutation burden differs between histological subtype of BE, perhaps reflecting in part the differing epigenetic stimuli from the environment such as acid and bile, as well as the histological organisation and topography of the stem cell gland unit. Patches of adjacent glands can share the same mtDNA mutation, indicating that glands spread by fission. Finally we have shown the both glandular and squamous tissue can be derived from a common progenitor cell. Taken together these findings indicate that distinct oesophageal lineages may be topographically and developmentally interconnected making eradication of some oesophageal squamous lineages necessary in the prevention of EA.

OC-030 Barrett’S Epithelium Shows Evidence of Gastric and Intestinal Differentiation Programmes but Preserves the Proliferative and Stem Cell Architecture of Gastric Glands

Introduction The origin and development of Barrett’s oesophagus has long been discussed, with three main proposals for the origin of metaplasia: from oesophageal squamous epithelium, from upward migration of cardiac glands, or from submucosal glands. Methods In Barrett’s glands we have studied the distribution of proliferative activity using Ki67 and the migration of cells in Barrett’s glands from patients infused with iododeoxyuridine 7 and 11 days pre-oesophagectomy. We have localised gene expression of mucins using immunohistochemistry (IHC) and trefoil family factor (TFF) peptides using IHC and in situ hybridization (ISH) and the stem cell marker Lgr5 using ISH, combining this with analysis of the clonal architecture of Barrett’s glands using mitochondrial DNA (mtDNA) as a clonal marker. Results In Barrett’s glands proliferation is seen mainly in the middle part of the gland and diminishes towards the surface and the base of the gland. Cells migrate in a bidirectional manner. MUC5AC and TFF1 expression are found superficially, while MUC6 and TFF2 are found at the bases of the glands, similar to the distribution seen in antral gastric glands: MUC2, staining goblet cells and columnar cells, is concentrated superficially. Lgr5 mRNA is also found in the middle part of the glands, indicating the location of the stem cell niche. Barrett’s glands are clonal, indicating derivation from a single cell, and suggesting that Barrett’s stem cells have dual differentiation capacity. Gastric intestinal metaplasia, on the other hand, shows basal Lgr5 mRNA localisation with a distribution of proliferative activity similar to intestinal crypts. Conclusion We conclude that Barrett’s glands show the proliferative and stem cell architecture, and preserve patterns of gene expression of pyloric-type gastric glands, but are maintained by unique stem cells with both gastric and intestinal differentiation capacity: we propose that Barrett’s glands originate as gastric glands and that subsequent intestinal differentiation advances with time, strongly supporting an origin from the proximal cardiac mucosa. Disclosure of Interest None Declared

OC-065 Stem cell analysis by label retention within Barrett's oesophagus

Introduction Stem cells are thought to be the only long lived cells in the gastrointestinal epithelium. It is thought they are the target for mutations that may lead to the premalignant Barretts Metaplasia (BM). These cells are thought to divide slowly allowing DNA dyes to be retained within the stem cells over a long period of time. Here, we have used this characteristic to identify, for the first time, the location of stem cells within the glands of Barretts oesophagus, an important premalignant condition. Methods We utilised iododeoxyuridine (IUdR) a thymidine analogue which is incorporated into the DNA of replicating cells, as a marker to identify the label retaining cells (LRC) as putative stem cells. Four patients with BM diagnosed with oesophageal adenocarcinoma undergoing oesophagectomy were infused with IudR prior to their surgery (7–69 days). After resection, tissue samples of normal oesophagus, BM, normal stomach and tumour were extracted for immunocytochemistry and FACS. Results Infusion 3 days or less in vitro in transformed cells and explants revealed appropriate abundant staining of the proliferative compartments. Results from FACS analysis showed the percentage of BRdU labelled cells was much greater in BM tissue (7.85%) compared with fundus (0.69%) or squamous (0.38%). However labelling at 7 days in both patients ex vivo showed positive discrete staining of LRCs within various gastrointestinal tissue types including BM. LRCs were seen in the basal layer of the squamous epithelium, likewise LRCs were seen at the neck/foveolar regions of the gastric tissue conforming to the suggested gastric SC zone. LRCs were seen at several locations in metaplastic tissue both at the base of the gland and in the neck region at 7, 11, 29 and 67 days, this ties in with preliminary data showing LGR5 (a stem cell marker) is also expressed at the base of the Barretts gland. Conclusion This is the first report of LRCs within the human oesophagus and our findings correlate with the hypothesised location of SCs in the squamous epithelium and gastric mucosa. Furthermore LRCs were located in the base of the BM gland implying that this is the location of BM SCs. Analysis and molecular characterisation of LRCs and BM SCs and their niche are underway, involving the use of other GI SC markers.