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Individual crypt genetic heterogeneity and the origin of metaplastic glandular epithelium in human Barrett's oesophagus
  1. Simon J Leedham (simon.leedham{at}cancer.org.uk)
  1. Cancer Research UK, United Kingdom
    1. Sean L Preston (seanlpreston{at}googlemail.com)
    1. St Barts and Royal London School of Medicine and Dentistry, United Kingdom
      1. Stuart AC McDonald (stuart.mcdonald{at}cancer.org.uk)
      1. Cancer Research UK, United Kingdom
        1. George Elia (george.elia{at}cancer.org.uk)
        1. Cancer Research UK, United Kingdom
          1. Pradeep Bhandari (pradeep.bhandari{at}porthosp.nhs.uk)
          1. Queen Alexandra Hospital, United Kingdom
            1. David Poller (david.poller{at}porthosp.nhs.uk)
            1. Queen Alexandra Hospital, United Kingdom
              1. Rebecca Harrison (rebecca.harrison{at}uhl-tr.nhs.uk)
              1. Leicester General Hospital, United Kingdom
                1. Marco R Novelli (m.novelli{at}ucl.ac.uk)
                1. University College Hospital London, United Kingdom
                  1. Janusz A Jankowski (janusz.jankowski{at}clinical-pharmacology.oxford.ac.uk)
                  1. University of Oxford, United Kingdom
                    1. Nicholas A Wright (warden{at}qmul.ac.uk)
                    1. Cancer Research UK, United Kingdom

                      Abstract

                      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 micro-dissecting 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 micro-dissection 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, that 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 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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