Research Article: Extensive telomere erosion is consistent with localised clonal expansions in Barrett’s metaplasia

Date Published: March 31, 2017

Publisher: Public Library of Science

Author(s): Boitelo T. Letsolo, Rhiannon E. Jones, Jan Rowson, Julia W. Grimstead, W. Nicol Keith, Gareth J. S. Jenkins, Duncan M. Baird, Arthur J. Lustig.


Barrett’s oesophagus is a premalignant metaplastic condition that predisposes patients to the development of oesophageal adenocarcinoma. However, only a minor fraction of Barrett’s oesophagus patients progress to adenocarcinoma and it is thus essential to determine bio-molecular markers that can predict the progression of this condition. Telomere dysfunction is considered to drive clonal evolution in several tumour types and telomere length analysis provides clinically relevant prognostic and predictive information. The aim of this work was to use high-resolution telomere analysis to examine telomere dynamics in Barrett’s oesophagus. Telomere length analysis of XpYp, 17p, 11q and 9p, chromosome arms that contain key cancer related genes that are known to be subjected to copy number changes in Barrett’s metaplasia, revealed similar profiles at each chromosome end, indicating that no one specific telomere is likely to suffer preferential telomere erosion. Analysis of patient matched tissues (233 samples from 32 patients) sampled from normal squamous oesophagus, Z-line, and 2 cm intervals within Barrett’s metaplasia, plus oesophago-gastric junction, gastric body and antrum, revealed extensive telomere erosion in Barrett’s metaplasia to within the length ranges at which telomere fusion is detected in other tumour types. Telomere erosion was not uniform, with distinct zones displaying more extensive erosion and more homogenous telomere length profiles. These data are consistent with an extensive proliferative history of cells within Barrett’s metaplasia and are indicative of localised clonal growth. The extent of telomere erosion highlights the potential of telomere dysfunction to drive genome instability and clonal evolution in Barrett’s metaplasia.

Partial Text

Barrett’s oesophagus is an acquired, hyper-proliferative and premalignant lesion that arises as a result of prolonged chronic gastroesophageal reflux disease. It leads to the metaplastic replacement of the squamous lining of the lower oesophagus by columnar intestinal-like epithelium and goblet cells [1, 2] and predisposes to the development of oesophageal adenocarcinoma [3, 4]. Barrett’s oesophagus is characterised by genetic heterogeneity; including large-scale copy number changes across the genome, with key loss of heterozygosity (LOH) events at 9p (involving the CDKN2A locus) and 17p (the TP53 locus) that facilitate progression [5, 6]. The loss of TP53 is also considered permissive for the subsequent development of aneuploidy and tetraploidy [7]. This genetic heterogeneity provides the diversity upon which clonal selection can operate and drive progression to adenocarcinoma [8]. The mechanisms that underpin the genetic heterogeneity observed in Barrett’s oesophagus have not been formally identified.

The loss of loci on 17p (p53), 11q (cyclin D1) and 9p (p16) are known to be related to histological progression in Barrett’s oesophagus [5, 6, 32], we considered that chromosome specific telomere dynamics may drive the loss of loci on specific chromosome arms in Barrett’s oesophagus. To investigate this we examined the telomere length distributions at the chromosome ends of 17p, 11q and 9p, together with XpYp, a chromosome arm that has not been documented to suffer copy-number changes in Barrett’s oesophagus. Analysis was undertaken using the highly sensitive single telomere length analysis (STELA) technique, that is capable of determining the telomere length of specific chromosome ends, and importantly can detect the very short telomeres that are capable of undergoing fusion; these telomeres are not represented in other telomere-length assays [18, 19]. We analysed a small cohort of patients (n = 8) with Barrett’s oesophagus, from which matched samples of gastric mucosae, Barrett’s metaplasia and squamous epithelia had been obtained (Fig 1A and 1B). Despite considerable variation in telomere length among individuals, each individual displayed similar patterns of the telomere length profiles in the tissues and at all four of the chromosome ends analysed (Fig 1A–1F). There was no trend for any one telomere to be significantly shorter than any of the others (p = .51; Fig 1G). It was clear from this analysis that the samples containing Barrett’s metaplasia, exhibited the shortest telomere length profiles in 7 of the 8 individuals analysed (Highlighted in green, Fig 1C–1F). Interestingly however, it was also apparent that in 3 of the patients analysed, the Gastric mucosa and Barrett’s metaplasia displayed indistinguishably short telomere length profiles (Fig 1C–1F). Moreover, the telomere length profiles exhibited substantial heterogeneity, with examples of apparent bimodal distributions observed in some samples, for example patient #2 (Fig 1A); these profiles are consistent with a heterogeneous cellular composition of the samples. Together these data indicate that no one specific telomere is likely to suffer preferential telomere erosion in BE, and whilst Barrett’s metaplasia exhibits telomere erosion, this can also be observed in normal gastric mucosae, with both showing shorter telomere lengths than squamous oesophagus.

Our data show that, compared to normal patient-matched squamous epithelium, tissues exhibiting Barrett’s metaplasia display significantly shorter telomeres at all the chromosomes tested. The extent of the difference was considerable with differences in mean telomere length of up to 4.5 kb. If one considers the telomere length profiles of the normal squamous epithelium to be representative of the progenitors from which the Barrett’s metaplasia was formed, then the large telomere length differentials observed are consistent with an extensive replicative history. The telomere dynamics and rates of erosion will be modulated by the replicative kinetics and telomerase activities in these tissues, as well as genotoxic insults that may drive stochastic telomeric shortening. It will thus be difficult to use the telomere length differential between normal and Barrett’s metaplasia to precisely estimate the amount of cell division. However in the absence of telomerase activity, assuming a telomere erosion rate of 85 bp/population doubling as observed in normal telomerase negative human fibroblast populations [33], differences of over 2kb between normal squamous epithelium and Barrett’s metaplasia would represent over 23 cell divisions. Clearly in the presence of telomerase that has been previously documented in Barrett’s metaplasia [31], the rates of telomere erosion will be less and thus the telomere length differentials will represent an even greater number of cell divisions. Moreover, the extent of telomere erosion was not consistent throughout the Barrett’s metaplasia segments, instead we observed distinct differences in both telomere length and the heterogeneity of the distributions within the different zones analysed. Telomere length heterogeneity reflects the clonal composition of the tissue analysed, with more homogeneous distributions indicative of clonal cell populations [33, 34]. Our data indicate that distinct clonal expansions can be observed at different positions throughout the Barrett’s Oesophagus segments and that this varies between patients. Taken together the observations of extensive telomere erosion and clonal expansion are consistent with an extensive replicative history of cells during the development of Barrett’s metaplasia [35].