Crypt dysplasia in Barrett's oesophagus shows clonal identity between crypt and surface cells

Epithelial dysplasia is an important histological diagnosis signifying the presence of pre‐invasive disease, usually needing intervention. However, the specific genetic changes responsible for the induction of this phenotypic change are unknown. Moreover, recent reports indicate that the dysplastic phenotype may not be immutable: in basal crypt dysplasia (CD), unequivocal dysplastic changes are seen in the crypts in Barrett's oesophagus and other pre‐invasive lesions in the gastrointestinal tract, but the upper crypts and surface epithelium associated with these dysplastic crypts show the definitive morphology of a differentiated epithelium. The genotypic relationship between CD and the differentiated surface epithelium is presently unclear. We obtained 17 examples of CD: the lower and upper crypts and surface epithelium were differentially laser‐microdissected from formalin‐fixed, paraffin‐embedded sections and mutations were sought in tumour suppressor genes frequently associated with progression in Barrett's oesophagus. We found two patients who both showed a c. C238T mutation in the CDKN2A (CDKN2AInk4A) gene and where the precise microanatomical relationships could be discerned: this mutation was present in both the CD at the crypt base and in the upper crypt and surface epithelium. We conclude that, in CD, the dysplastic basal crypt epithelium and the upper crypt and surface epithelium show clonal CDKN2A mutations, thus showing definitively that the surface epithelium is derived from the dysplastic crypt epithelium: the dysplastic phenotype is therefore not fixed and can be reversed. The mechanism of this change is unclear but may be related to the possibility that dysplastic cells can, probably early in their progression, respond to differentiation signals. However, it is also clear that a heavy mutational burden can be borne by crypts in the gastrointestinal tract without the development of phenotypic dysplasia. We are evidently some way from understanding the plasticity and the genotypic correlates of the dysplastic phenotype. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Dysplasia and early neoplasia in Barrett's oesophagus

Over the last 20–30 years, oesophageal adenocarcinoma has increased six-fold in the west, the majority complicating Barrett's. The greatest risk is associated with higher grades of dysplasia. Although there is ongoing research into molecular alterations, which may be helpful in predicting progression to cancer, the main predictive indicator remains the histological identification and grade of dysplasia. Significant inter and intraobserver variability in the diagnosis of dysplasia is well documented and atypia can be seen in other settings including inflammation. Given the screening and management implications for the patient, a robust diagnosis is essential, such that agreement between two pathologists with an interest in gastrointestinal pathology is of paramount importance, together with regular communication between pathologists and clinicians.     

Activation of Wnt signalling promotes development of dysplasia in Barrett's oesophagus

Barrett's oesophagus is a precursor of oesophageal adenocarcinoma, via intestinal metaplasia and dysplasia. Risk of cancer increases substantially with dysplasia, particularly high-grade dysplasia. Thus, there is a clinical need to identify and treat patients with early-stage disease (metaplasia and low-grade dysplasia) that are at high risk of cancer. Activated Wnt signalling is critical for normal intestinal development and homeostasis, but less so for oesophageal development. Therefore, we asked whether abnormally increased Wnt signalling contributes to the development of Barrett's oesophagus (intestinal metaplasia) and/or dysplasia. Forty patients with Barrett's metaplasia, dysplasia or adenocarcinoma underwent endoscopy and biopsy. Mice with tamoxifen- and β-naphthoflavone-induced expression of activated β-catenin were used to up-regulate Wnt signalling in mouse oesophagus. Immunohistochemistry of β-catenin, Ki67, a panel of Wnt target genes, and markers of intestinal metaplasia was performed on human and mouse tissues. In human tissues, expression of nuclear activated β-catenin was found in dysplasia, particularly high grade. Barrett's metaplasia did not show high levels of activated β-catenin. Up-regulation of Ki67 and Wnt target genes was also mostly associated with high-grade dysplasia. Aberrant activation of Wnt signalling in mouse oesophagus caused marked tissue disorganization with features of dysplasia, but only selected molecular indicators of metaplasia. Based on these results in human tissues and a mouse model, we conclude that abnormal activation of Wnt signalling likely plays only a minor role in initiation of Barrett's metaplasia but a more critical role in progression to dysplasia.     

p53 expression in Barrett's oesophagus,dysplasia, and adenocarcinoma using antibody DO-7

Barrett's oesophagus has a well-recognized association with oesophageal adenocarcinoma, with phenotypic progression through dysplasia to malignancy. The nuclear phosphoprotein p53 is a putative tumour suppressor with mutations resulting in both loss of negative growth regulatory function and possible gain of oncogene function. Many mutant forms have a prolonged half-life and are demonstrable with immunohistochemical techniques. We examined 62 endoscopic oesophageal biopsies and 36 oesophageal resections for p53 overexpression using the monoclonal antibody DO-7 on paraffin-embedded tissue. The series included 40 cases of Barrett's metaplasia, 13 cases of dysplasia, and 81 cases of adenocarcinoma. None of the cases of metaplasia was p53-positive, compared with 4/13 cases of dysplasia and 52/81 cases of adenocarcinoma. There was no association between the degree of dysplasia and p53 expression, although a trend emerged of increasing p53 expression with higher tumour grade. We conclude that p53 overexpression is frequent in oesophageal adenocarcinoma and may be related to tumour grade. p53 overexpression is not restricted to neoplastic lesions and mutation of this tumour suppressor may occur early in the malignant progression of Barrett's oesophagus.     

Strategies for identifying dysplasia in Barrett's oesophagus

Early neoplastic changes in Barrett's oesophagus are often only recognizable visually as discrete mucosal irregularities. With timely diagnosis, the prognosis is excellent, in contrast to advanced tumours in Barrett's oesophagus. The international specialist societies therefore recommend regular endoscopic surveillance for patients who have been diagnosed with Barrett's oesophagus. Following the 1993 Seattle Protocol, the various guidelines consistently require four-quadrant biopsy sampling every 1–2 cm over the entire Barrett's segment and additional biopsies from visually suspicious-appearing areas. This approach is time-consuming and costly, and inevitably involves sampling errors. Constant improvements in the image quality of modern endoscopes in recent years have made a vital contribution to increasing the detection rate of early neoplastic changes in Barrett's oesophagus. The aim of new technological developments is to increase the detection rate further. This article reviews the current state of technological developments and assesses their value.     

In Brief: The (molecular) pathogenesis of Barrett's oesophagus

Barrett's oesophagus is a metaplastic change, such that the normal squamous epithelial lining of the oesophagus is replaced by specialized columnar‐lined epithelium. Barrett's oesophagus is clinically significant and has a high health economic impact as it is associated with heightened risk of progression to oesophageal adenocarcinoma. This review discusses the pathogenesis of Barrett's oesophagus with an emphasis on the underlying molecular events. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Apoptotic and proliferative activity in the neoplastic progression of Barrett's oesophagus: a comparative study

The balance between proliferation and apoptosis within a tissue is important in controlling its overall growth. When either or both are altered, uncontrolled cell proliferation can contribute to cancer. The aim of this study was to investigate apoptosis and proliferation in the progression from Barrett's oesophagus to adenocarcinoma. Fifty-one paraffin sections of Barrett's mucosa with both intestinal and gastric-type Barrett's mucosa, dysplasia, and adenocarcinoma, from 28 patients, were examined for apoptosis using haematoxylin and eosin (H&E)-stained sections counterstained immunohistochemically with CD45 to distinguish leucocytes from apoptotic bodies. Proliferation was detected by immunohistochemistry using the MIB-1 (Ki-67) antibody. There was an increase in proliferation in dysplastic and carcinomatous tissue compared with metaplastic tissue (p = 0·0001). In dysplasia, proliferation was distributed throughout the basal–luminal axis, whereas in metaplasia, cell division was compartmentalized to the lower crypt (p < 0·001). Conversely, there was a decrease in apoptosis in the upper crypt and luminal surface in dysplasia and adenocarcinoma compared with metaplasia (p < 0·0008). There was a significant increase in apoptotic activity in intestinal-type Barrett's mucosa compared with gastric-type. There was a highly significant increase in the glandular proliferation to apoptosis ratio (GPAR) in the progression of metaplasia to dysplasia to adenocarcinoma (p = 0·001). The shift in the GPAR in the progression of neoplastic change suggests that it may be a useful and sensitive marker of neoplastic change in Barrett's oesophagus, especially if the assessment of both apoptotic and proliferative activity in the mucosa can be made easier by more sophisticated technical methods. Copyright © 1999 John Wiley & Sons, Ltd.     

Regulatory peptides in Barrett's oesophagus

Barrett's epithelium refers to the presence of ectopic mucosal types in the squamous-lined oesophagus. Previous studies have documented argentaffin and argyrophil-positive cells as well as gastrin-like immunoreactivity in oesophageal tissue extracts from patients with Barrett's mucosa. In the present study, 125 oesophageal biopsies obtained under direct vision at endoscopy from 22 patients with Barrett's oesophagus were systematically studied using fluorescence and peroxidase antiperoxidase single and double-staining immunocytochemical methods employing highly specific antibodies to localize the following peptide-containing cell types in Barrett's mucosa: gastrin, somatostatin, gastric inhibitory polypeptide, motilin, neurotensin and pancreatic glucagon. In addition, EC cells were localized using a cytochemical silver staining method. The results of this study indicate that EC cells and gastrin- and somatostatin-containing endocrine cells are detectable in Barrett's epithelium.     

The changing role of the pathologist in the management of Barrett's oesophagus

Pathological specimens from columnar‐lined oesophagus (CLO) comprise a considerable proportion of the workload of gastrointestinal pathologists in Western countries. There remain controversies concerning the diagnostic role of pathology. More recently, in the UK at least, the diagnosis has been regarded as primarily an endoscopic endeavour, with pathology being corroborative and only diagnostic when endoscopic features are equivocal or when there are additional features that make the endoscopic diagnosis unclear. There is also recognition that demonstration of intestinalisation or ‘goblet cells’ is not paramount, and should not be required for the diagnosis. There have been notable changes in the management of CLO neoplasia: pathologists are centrally involved in its management. Pathological assessment of endoscopic mucosal resection (EMR) specimens provides the most useful means of determining the management of early neoplasia and of determining indications for surgery. This represents an extraordinarily rapid change in management, in that, <10 years ago, laborious Seattle‐type biopsy protocols were recommended, and high grade dysplasia was an indication for resectional surgery. Now, individual patient management is paramount: multi‐professional meetings determine management after biopsy and EMR assessment. One significant change is that major resections are undertaken less often, in Western countries, for CLO neoplasia.     

Ki67 and p53 immunohistochemistry reduces interobserver variation in assessment of Barrett's oesophagus

AIMS: To devise clinically applicable methods for assessing p53 and Ki67 immunohistochemical (IHC) reactivity in Barrett's oesophagus (BE) and to compare the interobserver agreement between these methods and routine haematoxylin and eosin (H&E) evaluation. METHODS AND RESULTS: One hundred and fifteen biopsies diagnosed as BE, selected from the files of the University Hospital MAS, Malmo, were re-evaluated for dysplasia by three pathologists. For IHC analysis areas with the most prominent positivity were evaluated. The mean of p53+ epithelial nuclei/high-power field (HPF) was obtained by counting between 1 and 5 HPFs/biopsy. A proliferation quotient (PQ) was obtained by dividing the number of Ki67+ epithelial nuclei in the upper half by the lower half of the mucosa, using two HPFs. Mean kappa values were 0.24, 0.71 and 0.52 for H&E, p53 and Ki67 evaluations, respectively. There was a correlation between increasing severity of dysplasia, IHC measurable overexpression of p53 and shift of the mucosal proliferation zone towards the surface, measured as PQ. CONCLUSIONS: The described methods for p53 and Ki67 evaluation are more reproducible than routine H&E evaluation of BE. Furthermore, the IHC methods correlate with the severity of dysplasia and are useful supplementary prognostic markers.     

Inter-observer variation between general and specialist gastrointestinal pathologists when grading dysplasia in ulcerative colitis

Histological dysplasia is the cornerstone of colorectal cancer surveillance in ulcerative colitis (UC). Recently, pathologists have received unfavourable media attention concerning other cancer screening programmes. The aim of this study was to determine whether colonic biopsy specimens should be examined by gastrointestinal pathologists as opposed to generalists, by examining inter-observer variation between the two groups. Fifty-one coded slides showing varying degrees of dysplasia were mailed to seven gastrointestinal and six general histopathologists. Pathologists allocated each biopsy into one of four categories without the benefit of a clinical history or an opportunity to use the 'indefinite' category that is included in the Riddell classification. The responses were analysed using kappa statistics. The overall kappa statistic for gastrointestinal pathologists was 0.30 [95% confidence interval (CI)=0.26-0.34] and for general pathologists 0.28 (95% CI=0.23-0.32). Agreement was best for high-grade dysplasia (kappa of 0.54 and 0.61 for GI and general pathologists, respectively). There was total concordance of the 13 pathologists in only four of the 51 slides (7.8%) (95% CI=0.4-15.2%). It is concluded from these results that gastrointestinal pathologists are no better than generalists when grading dysplasia in UC and that agreement is poor in both groups. There is therefore no evidence that there would be any benefit in having specialist histopathology centres concentrating specifically on the interpretation of all surveillance colonoscopy biopsies from around the UK. It must be made clear to the public that surveillance and screening programmes carry a significant rate of histological error and that perfection cannot be expected or achieved with present methods.     

Accumulation of p53 protein in normal,dysplastic, and neoplastic Barrett's oesophagus

Accumulation of p53 protein was determined by immunohistochemisty in archival material of biopsy specimens from 102 patients with Barrett's oesophagus with different grades of dysplasia, in 24 oesophageal adenocarcinomas associated with Barrett's oesophagus, and in 23 cases of metaplatic epithelium adjacent to these carcinomas. Immunostaining for the p53 protein was found in 23/102 (23 per cent) cases of the Barrett's oesophagus biopsies and in 12/23 (52 per cent) cases of Barrett's oesophagus adjacent to adenocarcinoma. Significant correlations were found between the grade of dysplasia and p53 immunoreactivity in both Barrett's biopsies without adenocarcinoma (P<0.001) and Barrett's oesophagus adjacent to adenocarcinoma (P<0.05). In the adenocarcinomas, intense nuclear immunohistochemical staining for p53 was diffusely or focally present in 20/24 (83 per cent) of the specimens. In Barrett's oesophagus, p53 is a progression marker with high expression in high-grade dysplasia (89 per cent) and adenocarcinoma (83 per cent).     

Distribution of laminins in the basement membranes of the upper gastrointestinal tract and Barrett's oesophagus

Barrett's oesophagus predisposes to oesophageal adenocarcinoma. In vitro, laminin, a component of the epithelial basement membrane (BM), is important in regulation of cell differentiation. There is limited information on the distribution of laminin chains in the upper gastrointestinal tract (GIT) and none in Barrett's oesophagus. This study aimed to investigate qualitatively the distribution of laminins in the normal upper GIT mucosa and Barrett's oesophagus in order to understand the role of laminins in metaplasia. Immunoperoxidase staining for laminin chains alpha1, alpha2, alpha3, alpha5, beta1, beta2, beta3, gamma1, and gamma2 was performed on frozen endoscopic squamous and Barrett's oesophageal biopsies and surgical resection specimens from squamous oesophagus (in resection specimens for oesophageal cancer), and in oesophageal and gastric biopsies from control subjects. alpha1 laminin was expressed in the BM of submucosal glands and ducts in squamous oesophagus and Brunner's glands in the duodenum, but not in Barrett's oesophagus or elsewhere in the upper GIT. alpha2 laminin chain was expressed in a granular distribution in the BM of squamous epithelium. In columnar epithelium, including Barrett's oesophagus, alpha2 laminin chain was expressed continuously in the BM of glands and deeper pits, but expression was reduced and granular in the surface epithelial BM. beta2 laminin was continuous in squamous epithelial BM, but in Barrett's and cardia, gastric body, and duodenum, it was expressed faintly in the surface but continuously in the BM of glands and deeper pits. The constituents of laminin-5 were continuously expressed in the BM of squamous epithelium, but in the cardia, gastric body, duodenum, and Barrett's, they were expressed only in the BM of surface epithelium, with a sharp decline in the glandular and deeper pit BM. Site-specific distribution of the alpha2 and beta2 laminin chains may therefore have an important role in Barrett's metaplasia. However, the absence of alpha1 laminin in Barrett's mucosa suggests that this is unlikely to play an important role in columnar metaplasia.     

The diagnosis of dysplasia and malignancy in Barrett's oesophagus

Barrett's metaplasia is associated with an increased risk for adenocarcinoma. Adenocarcinoma develops through a multistep process characterized by defects in genes and morphological abnormalities. The early morphological changes of the process are called 'dysplasia'. Dysplasia is defined as an unequivocal neoplastic (premalignant) transformation confined within the basement membrane. For most Western pathologists malignancy is defined as invasion and characterized by a breach through the basement membrane. Japanese pathologists rely on cytological atypia and complex branching of crypts. Cytological and architectural abnormalities allow identification of dysplasia on routinely stained sections. A distinction is made between low- and high-grade dysplasia. The differential diagnosis between low-grade dysplasia and reactive changes can be difficult. Therefore a second opinion is strongly recommended, not only for high-grade dysplasia but also for low-grade. Immunohistochemistry for p53 and flow cytometry for detection of aneuploidy can support the diagnosis. Identification of dysplasia and malignancy depends on the number of biopsy samples examined. The minimum number of biopsies required has not yet been determined and depends partly on the length of the metaplastic segment. It has been proposed to sample with four quadrant biopsies at 20-mm intervals. New endoscopic techniques can increase the diagnostic yield. Endoscopically visible lesions increase the risk of finding malignancy. The time sequence for the progression of dysplasia is not known but progression from low- to high-grade and cancer has been shown to occur over a period of years although it may not be inevitable.     

Barrett's oesophagus--a pathologist's view

Barrett's oesophagus, a precancerous condition for oesophageal adenocarcinoma, detected on endoscopy and confirmed on histology, shows intestinal metaplasia of the lower oesophagus. The significance of microscopic foci of intestinal metaplasia at the gastro-oesophageal junction, corresponding either to so-called 'ultrashort' segment Barrett's oesophagus, or to carditis with intestinal metaplasia, is still a matter of debate. The surveillance of patients with Barrett's oesophagus is still based on systematic biopsy sampling of Barrett's mucosa on endoscopy, looking for dysplasia. Although well-established classifications of dysplasia are now used by most pathologists, there remain numerous problems with this subjective marker (sampling, diagnostic reproducibility, natural history, etc). Therefore, many alternative biomarkers have been proposed, but only DNA aneuploidy, proliferation markers and p53 loss of heterozygosity/overexpression have been shown to be of some use at the present time. Some endoscopic improvements already allow a better selection of biopsies, and it may be that in future new technologies will allow 'virtual biopsies'. On the other hand, the role of pathologists now extends to the evaluation of new therapeutic modalities of early neoplastic lesions in Barrett's oesophagus, especially endoscopic mucosal resection.     

Molecular evaluation of ablative therapy of Barrett's oesophagus

Barrett's oesophagus is a major risk factor for developing oesophageal adenocarcinoma. Ablation by argon plasma coagulation (APC) and photodynamic therapy (PDT) is currently under investigation for the removal of metaplastic and dysplastic Barrett's oesophagus. This study examined the effect of ablative therapy on Barrett's oesophagus at cell-cycle and genetic levels. The premalignant potential of residual or recurring Barrett's oesophagus was assessed by p53 immunohistochemistry, Ki67-related proliferative capacity, and DNA ploidy status (ie an abnormal chromosome 1 number) as measured by interphase in situ hybridization. Twenty-nine patients with Barrett's oesophagus (23 male and 6 female, mean age 58 years, mean length of Barrett's oesophagus 4 cm) were treated with APC or PDT. Intestinal metaplasia without dysplasia was present in 16 patients, low-grade dysplasia in five, and high-grade dysplasia in eight patients. Biopsy samples were obtained at regular intervals (mean follow-up 20 months, range 6-36 months). One month after the first ablation, Barrett's oesophagus was no longer identified, either endoscopically or histologically, in nine patients (32%). At this time point, significant down-grading was achieved for abnormal chromosome 1 numbers (p = 0.020) and Ki67-defined proliferation (p = 0.002). Patients with residual Barrett's oesophagus were additionally treated with APC, resulting in the elimination of Barrett's oesophagus in 76% of all patients. However, at the last follow-up endoscopy, metaplasia without dysplasia was still present in five patients, and low- and high-grade dysplasia were each present in one patient. An abnormal chromosome 1 number and p53 protein overexpression were detected only in the high-grade dysplastic lesion, but increased proliferation was still present in the majority of these persisting cases. Although endoscopic removal of Barrett's oesophagus by ablative therapies is possible in the majority of patients, histologically complete elimination cannot be achieved in all cases. Persistent Barrett's oesophagus may still harbour molecular aberrations and must therefore be considered still to be at risk of progression to adenocarcinoma.