Histological assessment & use of immunohistochemical markers for detection of dysplasia in Barrett's esophageal mucosa.

BACKGROUND: Histological assessment of dysplasia in Barrett's esophagus (BE) has high inter-observer variability. Hence, use of ancillary markers for early detection of dysplasia in BE is an important clinical question. METHODS: In this retrospective study consecutive cases of BE (n = 59), over a period of 4 years were included. Hematoxylin and eosin stained sections were reviewed independently by 3 senior qualified pathologists, who graded the dysplasia according to the Vienna Classification system and inter-observer agreement was analysed using the Kappa statistics. Subsequently Alpha-Methyl Acyl-CoA Racemase (AMACR), p53, CyclinD1, ß-catenin, H2AX and M30 immunohistochemical (IHC) stains were examined on the following disease categories: BE with no dysplasia [NFD] (45), BE with indefinite for dysplasia (IFD) (4), low grade dysplasia (LGD) (3), high grade dysplasia (HGD) (2) and in adenocarcinomas (5). H score was calculated by adding up products of different grades of stain distribution and stain intensities (range of scores 0-300). RESULTS: Among the 3 pathologists, overall agreement was poor (k 0.06; 95% CI -0.089 to 0.145), with highest disagreement noted for differentiating the LGD and IFDs (k = 0.21). After revising the histological criteria, the kappa improved to 0.53. Among the IHC stains performed, p53, ß-catenin, H2AX and M30 stains were significantly useful to differentiate between IFD and LGD (P values: 0.04, 0.004, 0.05 & 0.04, respectively). AMACR and ß-catenin stains though were up-regulated in HGD/adenocarcinomas than in other categories, their expression were not statistically different between the IFD and LGDs. CONCLUSIONS: A detail histological scoring system may bring uniformity in histological interpretation of dysplasia in BE. Using a combined panel of IHC stains seems helpful in detection of dysplasia in BE, especially to differentiate the IFD and LGD changes in BE.

Immunohistochemical assessment of Survivin and Bcl3 expression as potential biomarkers for NF-κB activation in the Barrett metaplasia-dysplasia-adenocarcinoma sequence.

Non-dysplastic Barrett's oesophagus (NDBE) occurs as a consequence of an inflammatory response triggered through prolonged gastro-oesophageal reflux and it may precede the development of oesophageal adenocarcinoma. NF-κB activation as a result of the inflammatory response has been shown in NDBE, but the possible mechanism involved in the process is unknown. The aim of this study was to assess, using immunohistochemistry, Survivin and Bcl3 expression as potential biomarkers for NF-κB activation along the oesophageal metaplasia-dysplasia-adenocarcinoma sequence. Survivin is an NF-κB-inducible anti-apoptotic protein, and Bcl3 is a negative regulator of NF-κB. There was progressive upregulation of Survivin expression along the oesophageal metaplasia-dysplasia-adenocarcinoma sequence. Bcl3 expression was upregulated in non-dysplastic Barrett's oesophagus, low-grade, high-grade dysplasia and oesophageal adenocarcinoma when compared to squamous group. The study shows the differential expression of Bcl3 between the squamous and Barrett's stage, suggesting that Bcl3 could be a surrogate marker for early event involving constitutive NF-κB activation. In addition, the study suggests that NF-κB activation may infer resistance to apoptosis through the expression of anti-apoptotic genes such as Survivin, which showed progressive increase in expression throughout the oesophageal metaplasia-dysplasia-adenocarcinoma sequence. This ability to avoid apoptosis may underlie the persistence and malignant predisposition of Barrett's metaplasia.

Screening for Barrett's esophagus.

Barrett's esophagus is the only known esophageal precursor for the development of esophageal adenocarcinoma. However, screening for Barrett's esophagus remains controversial. Although screening is advocated in selected populations, it is unclear how it should be implemented. In this review, the current definition of Barrett's esophagus will be discussed. There will be a review of the emerging evidence supporting the cost-effectiveness of screening and surveillance for Barrett's esophagus in preventing esophageal adenocarcinoma. The known risk factors for Barrett's esophagus and the development of esophageal adenocarcinoma, currently utilized to determine the appropriate populations to screen, will be reviewed. Finally we will review the standard techniques utilized to screen for Barrett's esophagus and examine new technologies that might improve the efficacy and availability of Barrett's esophagus screening.

Gender difference in gastro-esophageal reflux diseases.

The incidence of esophageal adenocarcinoma (EAC) has risen sharply in western countries over the past 4 decades. This type of cancer is considered to follow a transitional process that goes from gastro-esophageal reflux disease (GERD) to Barrett's esophagus (BE, a metaplastic condition of the distal esophagus), a precursor lesion and ultimately adenocarcinoma. This spectrum of GERD is strongly predominant in males due to an unidentified mechanism. Several epidemiologic studies have described that the prevalence of GERD, BE and EAC in women is closely related to reproductive status, which suggests a possible association with the estrogen level. Recently, we revealed in an in vivo study that the inactivation of mast cells by the anti-inflammatory function of estrogen may account for the gender difference in the GERD spectrum. Other studies have described the contribution of female steroid hormones to the gender difference in these diseases. Estrogen is reported to modulate the metabolism of fat, and obesity is a main risk factor of GERDs. Moreover, estrogen could confer esophageal epithelial resistance to causative refluxate. These functions of estrogen might explain the approximately 20-year delay in the incidence of BE and the subsequent development of EAC in women compared to men, and this effect may be responsible for the male predominance. However, some observational studies demonstrated that hormone replacement therapy exerts controversial effects in GERD patients. Nevertheless, the estrogen-related endocrine milieu may prevent disease progression toward carcinogenesis in GERD patients. The development of innovative alternatives to conventional acid suppressors may become possible by clarifying the mechanisms of estrogen.

Evaluation of a minimally invasive cell sampling device coupled with assessment of trefoil factor 3 expression for diagnosing Barrett's esophagus: a multi-center case-control study.

BACKGROUND: Barrett's esophagus (BE) is a commonly undiagnosed condition that predisposes to esophageal adenocarcinoma. Routine endoscopic screening for BE is not recommended because of the burden this would impose on the health care system. The objective of this study was to determine whether a novel approach using a minimally invasive cell sampling device, the Cytosponge, coupled with immunohistochemical staining for the biomarker Trefoil Factor 3 (TFF3), could be used to identify patients who warrant endoscopy to diagnose BE. METHODS AND FINDINGS: A case-control study was performed across 11 UK hospitals between July 2011 and December 2013. In total, 1,110 individuals comprising 463 controls with dyspepsia and reflux symptoms and 647 BE cases swallowed a Cytosponge prior to endoscopy. The primary outcome measures were to evaluate the safety, acceptability, and accuracy of the Cytosponge-TFF3 test compared with endoscopy and biopsy. In all, 1,042 (93.9%) patients successfully swallowed the Cytosponge, and no serious adverse events were attributed to the device. The Cytosponge was rated favorably, using a visual analogue scale, compared with endoscopy (p < 0.001), and patients who were not sedated for endoscopy were more likely to rate the Cytosponge higher than endoscopy (Mann-Whitney test, p < 0.001). The overall sensitivity of the test was 79.9% (95% CI 76.4%-83.0%), increasing to 87.2% (95% CI 83.0%-90.6%) for patients with ≥3 cm of circumferential BE, known to confer a higher cancer risk. The sensitivity increased to 89.7% (95% CI 82.3%-94.8%) in 107 patients who swallowed the device twice during the study course. There was no loss of sensitivity in patients with dysplasia. The specificity for diagnosing BE was 92.4% (95% CI 89.5%-94.7%). The case-control design of the study means that the results are not generalizable to a primary care population. Another limitation is that the acceptability data were limited to a single measure. CONCLUSIONS: The Cytosponge-TFF3 test is safe and acceptable, and has accuracy comparable to other screening tests. This test may be a simple and inexpensive approach to identify patients with reflux symptoms who warrant endoscopy to diagnose BE.

Immunohistochemical assessment of NY-ESO-1 expression in esophageal adenocarcinoma resection specimens.

AIM: To assess NY-ESO-1 expression in a cohort of esophageal adenocarcinomas. METHODS: A retrospective search of our tissue archive for esophageal resection specimens containing esophageal adenocarcinoma was performed, for cases which had previously been reported for diagnostic purposes, using the systematised nomenclature of human and veterinary medicine coding system. Original haematoxylin and eosin stained sections were reviewed, using light microscopy, to confirm classification and tumour differentiation. A total of 27 adenocarcinoma resection specimens were then assessed using immunohistochemistry for NY-ESO-1 expression: 4 well differentiated, 14 moderately differentiated, 4 moderate-poorly differentiated, and 5 poorly differentiated. RESULTS: Four out of a total of 27 cases of esophageal adenocarcinoma examined (15%) displayed diffuse cytoplasmic and nuclear expression for NY-ESO-1. They displayed a heterogeneous and mosaic-type pattern of diffuse staining. Diffuse cytoplasmic staining was not identified in any of these structures: stroma, normal squamous epithelium, normal submucosal gland and duct, Barrett's esophagus (goblet cell), Barrett's esophagus (non-goblet cell) and high grade glandular dysplasia. All adenocarcinomas showed an unexpected dot-type pattern of staining at nuclear, paranuclear and cytoplasmic locations. Similar dot-type staining, with varying frequency and size of dots, was observed on examination of Barrett's metaplasia, esophageal submucosal gland acini and the large bowel negative control, predominantly at the crypt base. Furthermore, a prominent pattern of apical (luminal) cytoplasmic dot-type staining was observed in some cases of Barrett's metaplasia and also adenocarcinoma. A further morphological finding of interest was noted on examination of haematoxylin and eosin stained sections, as aggregates of lymphocytes were consistently noted to surround submucosal glands. CONCLUSION: We have demonstrated for the first time NY-ESO-1 expression by esophageal adenocarcinomas, Barrett's metaplasia and normal tissues other than germ cells.

Immunohistochemical assessment for Cdx2 expression in the Barrett metaplasia-dysplasia-adenocarcinoma sequence.

BACKGROUND: Barrett oesophagus (BO) occurs as a consequence of prolonged gastro-oesophageal reflux and it may precede the development of oesophageal adenocarcinoma. AIMS: The aim of this study was to accurately assess Cdx2 expression in the oesophageal metaplasia-dysplasia-adenocarcinoma sequence. METHOD: The expression of the transcription factor Cdx2 in Barrett metaplasia, high-grade glandular dysplasia and adenocarcinoma was investigated using immunohistochemistry. RESULTS: The results confirmed previous immunohistochemistry and PCR-based investigations, indicating that Cdx2 was expressed by intestinal metaplasia in the oesophagus. In addition, upregulation followed by linear downregulation of Cdx2 expression through the oesophageal metaplasia-dysplasia-adenocarcinoma sequence was demonstrated for the first time. CONCLUSION: Such downregulation of Cdx2 expression could be in keeping with a role as a tumour suppressor gene, but a simpler explanation would be downregulation of a differentiation marker.

Surveillance in Barrett's esophagus: a failed premise.

BACKGROUND: It is recommended that patients in whom Barrett's esophagus is diagnosed undergo surveillance endoscopy. However, multiple issues regarding the efficacy and feasibility of surveillance remain. METHODS: Quantitative techniques were used to examine surveillance in patients with Barrett's esophagus. A retrospective case-control study was performed to determine whether surveillance endoscopy prolonged survival in a cohort of U.S. veterans diagnosed with esophageal adenocarcinoma. Cost-effectiveness analysis was employed to compare competing strategies of management for patients with Barrett's esophagus to determine whether surveillance strategies using alternative biomarkers could out-perform dysplasia based surveillance, and whether new techniques for eradicating Barrett's metaplasia would constitute cost-effective strategies. RESULTS: Surveillance did not improve long-term survival among veterans diagnosed with esophageal adenocarcinoma. Lead-time bias has confounded previous reports claiming the efficacy of endoscopic surveillance. Cost-effectiveness analysis revealed that while screening 50-year old Caucasian males with heartburn may be cost-effective, surveillance even at 5 year intervals among patients with Barrett's esophagus without dysplasia exceeded the threshold of cost-effective care. If a biomarker were developed whose sensitivity and specificity to predict cancer development exceeded 80%, this could represent a more viable strategy than dysplasia-based surveillance and overcome the inherent inter- and intra-observer variations in dysplasia diagnosis that currently limit the effectiveness of surveillance programs. Finally, techniques that reduce cancer incidence such as endoscopic mucosal resection or ablation will likely be more cost-effective than current surveillance strategies that rely on early detection of cancer. CONCLUSIONS: Current recommendations for the management of patients with Barrett's esophagus are flawed. Future guidelines should include alternative markers of cancer risk and focus on strategies that reduce cancer incidence instead of cancer detection.

Paget cells in the esophagus: assessment of their histopathologic features and near-universal association with underlying esophageal adenocarcinoma.

Pagetoid spread of primary esophageal melanomas and several cases of pagetoid esophageal squamous cell carcinoma are known. However, true esophageal Paget disease (intraepithelial growth of neoplastic cells with glandular differentiation) has only rarely been reported. We encountered 3 endoscopic biopsy specimens containing Paget cells in squamous epithelium associated with adenocarcinomas in Barrett esophagus (BE) and in the esophagogastric junction. To determine the prevalence of Paget cells in the esophagus, we studied 81 endoscopic mucosal resections and 27 esophagectomies from patients with invasive or intramucosal adenocarcinoma, and compared the findings to a control group of 47 endoscopic mucosal resections and 25 esophagectomies from patients with high-grade dysplasia in BE. Paget cells were present in squamous epithelium overlying 5 (4.9%) of 108 adenocarcinomas but in none (0%) of 72 BE with high-grade dysplasia (P=0.16). A computerized search for primary Paget disease using the terms "Paget's and esophagus" or "pagetoid and esophagus" from 1994 to 2007 did not yield any additional cases. Among the 8 patients with Paget cells (including the 2 index biopsies) there were no differences in either sex distribution (7M:1F) or age (mean 62.4 y) as compared with 103 adenocarcinomas without Paget cells (93M:10F, P=0.58; mean age 69.2 y, P=0.78). Morphologically, all adenocarcinomas with Paget cells contained at least a component of diffuse, poorly differentiated adenocarcinoma, and 1 was a signet ring cell carcinoma. Paget cells involved only squamous epithelium directly above the poorly differentiated tumor foci. Histochemistry for periodic acid-Schiff with diastase (PAS-D) and mucicarmine, and immunohistochemistry for CK7, CK20, p53, and E-cadherin, were performed on 7 Paget cases with the following results: PAS-D+ (7 of 7, 100%), mucicarmine+ (6 of 7, 86%), CK7+ (7 of 7, 100%), CK20+ (5 of 7, 71%), p53 overexpression (3 of 7, 43%), and E-cadherin loss (complete loss in 1 and faint expression in 3, 57%). Overall, PAS-D was the most efficacious stain for highlighting Paget cells. A control group of 19 adenocarcinomas without Paget cells were also stained for E-cadherin; only 1 showed faint expression (5%) and none showed complete loss (P=0.01). These results demonstrate a low but significant prevalence (4.9%) of Paget cells in esophageal and esophagogastric junction adenocarcinomas. Unlike previously described cases of mammary, vulvar, and perianal Paget disease, esophageal Paget cells are almost universally associated with underlying adenocarcinoma and not with high grade dysplasia ("in situ" disease) or primary Paget disease. A commonality among cases with Paget cells is the presence of focal or diffuse, poorly differentiated adenocarcinoma with discohesive cells. E-cadherin alterations seem to play a less significant role.

COX-2, CDX2, and CDC2 immunohistochemical assessment for dysplasia-carcinoma progression in Barrett's esophagus.

BACKGROUND: Immunohistochemical changes associated with development of cancer in Barrett's esophagus offer potential areas of intervention to prevent and manage esophageal cancer. AIMS: To assess the role of cyclooxygenase 2, caudal-type homeobox transcription factor 2 and cell division cycle 2/cyclin-dependent kinase 1 in the Barrett's metaplasia-dysplasia-adenocarcinoma sequence. PATIENTS AND METHODS: Specimens from 46 patients with Barrett's esophagus (39% without dysplasia, 33% with dysplasia and 28% with adenocarcinoma) were stained for cyclooxygenase 2, caudal-type homeobox transcription factor 2 and cell division cycle 2. RESULTS: Cyclooxygenase 2: No expression differences between groups were found, except for adenocarcinomas (p=0.04). Caudal-type homeobox transcription factor 2: Nuclear positivity decreased from Barrett's esophagus without dysplasia (71.6%), to Barrett's esophagus with low grade dysplasia (35.3%), to Barrett's esophagus with high grade dysplasia (17.14%); in adenocarcinoma these percentages were intermediate between high and low grade dysplasia (30.5%). Cell division cycle 2: Expression on deeper glandular structures was 40% in Barrett's esophagus without dysplasia, 55.47% in Barrett's esophagus with dysplasia, and 63.84% in adenocarcinoma, with no statistical differences between groups. Concerning cells of the superficial layer, Barrett's esophagus with low grade dysplasia expressed focal positivity (p=0.0001 vs. no dysplasia); Barrett's esophagus with high grade dysplasia displayed diffuse positivity (p=0.0001 vs. no dysplasia and low grade dysplasia). A diffuse positivity was found in Barrett's esophagus with adenocarcinoma (p=0.0001 vs. no dysplasia and low grade dysplasia). CONCLUSIONS: Further evaluation of cyclooxygenase 2, cell division cycle 2 and caudal-type homeobox transcription factor 2, in association with morphology, might help to improve the accuracy of diagnosis and be useful for the clinical-pathological assessment of patients with Barrett's esophagus.

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.

Assessment of proliferating cell nuclear antigen expression in dysplastic intestinal type Barrett's esophagus. Gruppo Operativo per lo Studio delle Precancerosi Esofagee.

In the management of patients with Barrett's Esophagus (BE) the definition of a sub-group of patients at high risk for malignant transformation of the metaplastic epithelium is important. Undoubtedly, the histological evaluation of dysplasia is the most important and objective parameter to identify a malignant transformation of BE. In order to obtain additional data a group of cell proliferation markers was applied in dysplastic lesions. We studied PCNA in a selected group of patients with BE. One hundred six biopsies were examined, referring to fifty five patients with BE, who were followed up for a period of at least one year (1-5 years). All patients showed one or more biopsies positive for dysplasia. PCNA was detected immunohistochemically on formalin fixed and paraffin embedded biopsies, and positive cells were evaluated in a crude percent count. Our results showed a remarkable number of PCNA positive cells in high grade dysplasia and a variable amount of PCNA positive cells in the others groups. In addition, there was an overlap in the number of positive cases between low grade dysplasia (30%), indefinite (33%) and negative (34%). Therefore, the assessment of S-phase cells correlated with the degree of dysplasia seems to be of limited practical use. Cell proliferation is probably affected by many factors and mainly inflammation.