Oesophagus

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

S J Leedham¹, S L Preston^1,2, S A C McDonald^1,3, G Elia¹, P Bhandari⁴, D Poller⁵, R Harrison⁶, M R Novelli⁷, J A Jankowski^1,3,8, N A Wright^1,2

¹ Histopathology Unit, Cancer Research UK, London, UK
² Institute of Cell and Molecular Sciences, St Bartholomew’s and Royal London School of Medicine and Dentistry, Queen Mary University, London, UK
³ Department of Clinical Pharmacology, University of Oxford, Oxford, UK
⁴ Department of Gastroenterology Queen Alexandra Hospital, Portsmouth, UK
⁵ Pathology Department, Queen Alexandra Hospital, Portsmouth, UK
⁶ Pathology Department, Leicester General Hospital, Leicester, UK
⁷ Histopathology Department, University College Hospital, London, UK
⁸ Digestive Disease Centre, Leicester Royal Infirmary, Leicester, UK

Dr Simon J Leedham, Histopathology Unit, Cancer Research UK, 44 Lincoln’s Inn Fields, London WC2A 3PX, UK; simon.leedham{at}cancer.org.uk

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.

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