A biomarker panel predicts progression of Barrett's esophagus to esophageal adenocarcinoma.

Progression from Barrett's esophagus (BE) to esophageal adenocarcinoma (EAC) is uncommon but the consequences are serious. Predictors of progression are essential to optimize resource utilization. This study assessed the utility of a promising panel of biomarkers applicable to routine paraffin embedded biopsies (FFPE) to predict progression of BE to EAC in a large population-based, nested case-control study.We utilized the Amsterdam-based ReBus nested case-control cohort. BE patients who progressed to high-grade dysplasia (HGD)/EAC (n = 130) and BE patients who never progressed (n = 130) were matched on age, sex, length of the BE segment, and duration of endoscopic surveillance. All progressors had minimum 2 years of endoscopic surveillance without HGD/EAC to exclude prevalent neoplasia. We assessed abnormal DNA content, p53, Cyclin A, and Aspergillus oryzae lectin (AOL) in FFPE sections. We performed conditional logistic regression analysis to estimate odds ratio (OR) of progression based on biomarker status.Expert LGD (OR, 8.3; 95% CI, 1.7-41.0), AOL (3 vs. 0 epithelial compartments abnormal; OR, 3.6; 95% CI, 1.2-10.6) and p53 (OR, 2.3; 95% CI, 1.2-4.6) were independently associated with neoplastic progression. Cyclin A did not predict progression and DNA ploidy analysis by image cytometry was unsuccessful in the majority of cases, both were excluded from the multivariate analysis. The multivariable biomarker model had an area under the receiver operating characteristic curve of 0.73.Expert LGD, AOL, and p53 independently predict neoplastic progression in BE patients and are applicable to routine practice. These biomarkers can aid in selecting patients for endoscopic ablation or more intensive surveillance.

Pilot study of cytological testing for oesophageal squamous cell dysplasia in a high-risk area in Northern Iran.

BACKGROUND: Oesophageal squamous cell carcinoma (ESCC) is a fatal disease with 5-year survival rates of <5% in Northern Iran. Oesophageal squamous dysplasia (ESD) is the precursor histologic lesion of ESCC. This pilot study was conducted to assess the feasibility, safety, and acceptability of non-endoscopic cytological examination of the oesophagus and to provide initial data on the accuracy of cytological atypia for identifying patients with ESD in this very-high-risk area. METHODS: Randomly selected asymptomatic participants of the Golestan Cohort Study were recruited. A cytological specimen was taken using a capsule sponge device and evaluated for atypical cells. Sections of the cytological specimen were also stained for p53 protein. Patient acceptability was assessed using a visual analogue scale. The cytological diagnosis was compared with a chromoendoscopic examination using Lugol's solution. RESULTS: Three hundred and forty-four subjects (43% male, mean (s.d.) age 55.6 (7.9) years) were referred to the study clinic. Three hundred and twelve met eligibility criteria and consented, of which 301 subjects (96.5%) completed both cytological and endoscopic examinations. There were no complications. Most of the participants (279; 92.7%) were satisfied with the examination. The sensitivity and specificity of the cytological examination for identifying subjects with high-grade ESD were 100 and 97%, respectively. We found an accuracy of 100% (95% CI=99-100%) for a combination of cytological examination and p53 staining to detect high-grade ESD. CONCLUSIONS: The capsule sponge methodology seems to be a feasible, safe, and acceptable method for diagnosing precancerous lesions of the oesophagus in this population, with promising initial accuracy data for the detection of high-grade ESD.

Endoscopic TriModal imaging and biomarkers for neoplasia conjoined: a feasibility study in Barrett's esophagus.

In Barrett's esophagus (BE), the normal squamous lining of the esophagus is replaced by specialized columnar epithelium. Endoscopic surveillance with autofluorescence imaging (AFI) and molecular biomarkers have been studied separately to detect early neoplasia (EN) in BE. The combination of advanced-imaging modalities and biomarkers has not been investigated; AFI may help detecting biomarkers as a risk-stratification tool. We retrospectively evaluated a cohort of patients undergoing endoscopy for EN in BE with AFI and correlated five biomarkers (HPP1, RUNX3, p16, cyclin A, and p53) in tissue samples with AFI and dysplasia status. Fifty-eight samples from a previous prospective study were selected: 15 true-positive (TP: AFI-positive, EN), 21 false-positive (FP: AFI-positive, no EN), 12 true-negative (TN1; AFI-negative, no EN in sample), 10 true-negative (TN2: AFI-negative, no EN in esophagus). Methylation-specific RT-PCR was performed for HPP1, RUNX3, p16, and immunohistochemistry for cyclin A, p53. P < 0.05 was considered statistically significant. Bonferroni correction was used for multiple comparisons. P16, cyclin A, p53 correlated with dysplasia (P < 0.01, P = 0.003, P < 0.001, respectively). Increased p16 methylation was observed between TP versus TN2 (P = 0.003) and TN1 versus TN2 (P = 0.04) subgroups, suggesting a field defect. Only p53 correlated with AFI-status (P = 0.003). After exclusion of EN samples, significance was lost. Although correlation with dysplasia status was confirmed for p16, cyclin A and p53, underlining the importance of these biomarkers as an early event in neoplastic progression, none of the investigated biomarkers correlated with AFI status. A larger prospective study is needed to assess the combination of AFI and a larger panel of biomarkers to improve risk stratification in BE.

Non-endoscopic screening biomarkers for Barrett's oesophagus: from microarray analysis to the clinic.

BACKGROUND AND AIMS: Barrett's oesophagus predisposes to oesophageal adenocarcinoma but the majority of patients are undiagnosed. A novel non-endoscopic cytological screening device, called a capsule sponge, makes population-based screening for the disease a feasible option. However, due to the mixed cell population retrieved by the capsule sponge, biomarkers specific for Barrett's oesophagus are required. METHODS: Three publically available microarray datasets were used to identify putative biomarkers present in Barrett's oesophagus but absent from normal oesophagus and gastric mucosa. Validation was performed by qPCR (n = 10 each of normal oesophagus, Barrett's oesophagus, gastric mucosa) and immunohistochemistry (normal oesophagus, n = 20; Barrett's oesophagus, n = 21; gastric mucosa, n = 24; duodenum, n = 18). The biomarker was then prospectively evaluated on capsule sponge specimens from 47 patients with Barrett's oesophagus and 99 healthy controls. RESULTS: 2/14 genes identified, dopa decarboxylase (DDC) and Trefoil factor 3 (TFF3), were confirmed by qPCR to be upregulated in Barrett's oesophagus compared to normal oesophagus (p<0.01) and gastric mucosa (p<0.01 and p<0.05, respectively). Immunohistochemistry confirmed that DDC protein expression was restricted to Barrett's oesophagus but was confined to <1% of the cells within the crypt compartment. TFF3 protein was expressed to high levels at the luminal surface of Barrett's oesophagus compared to absent expression in normal oesophagus and gastric mucosa (p<0.001). Using the capsule sponge 36/46 patients with Barrett's oesophagus (one inadequate sample) and 6/96 controls were positive for TFF3 giving a sensitivity of 78% and a specificity of 94%. CONCLUSIONS: TFF3 is a promising marker for Barrett's oesophagus screening since it is expressed at the luminal surface of Barrett's oesophagus but not in adjacent tissue types and may be applied to a non-endoscopic screening device.

Physiological and molecular analysis of acid loading mechanisms in squamous and columnar-lined esophagus.

SUMMARY: Barrett's esophagus (BE) may be an adaptive cellular response to repeated acid exposure. The aims of this study were to compare intracellular acid loading in BE cells with normal squamous esophageal cells. Primary squamous and BE cells were obtained endoscopically and cultured for up to 24 h. Barrett's adenocarcinoma cell lines TE7 and OE-33 were compared with a normal esophageal (NE) cell line OE-21. Extracellular pH was lowered to 6.0 using HCl; specific ion exchangers were blocked pharmacologically and pH microfluorimetry was performed using 2'7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. The effect of prolonged acid preincubations and repeated acid exposure on acid loading and recovery were examined. Acid loading was greater in primary BE than NE cells (DeltapHi -0.22 +/- 0.08 vs.-0.13 +/- 0.01) and maximal in the BE carcinoma cell line TE7 (DeltapHi -0.30 +/- 0.01). Whereas TE7 cells were able to recover fully from repeated acid exposure, OE-21 cells remained profoundly acidic. BE primary and transformed cells utilize DIDS inhibitable sodium-independent chloride/bicarbonate exchange as well as sodium/hydrogen ion exchange for acid loading. In contrast, SE only requires sodium-independent chloride/bicarbonate exchange for acidification. The degree of acid loading is greater in BE than NE cells and it occurs via dual ion exchangers similar to gastric mucosa. Only Barrett's epithelial cells can maintain a physiological pHi following prolonged and repeated reflux exposure, which may confer a teleological advantage.

Histopathological and molecular analysis of gastrectomy specimens from hereditary diffuse gastric cancer patients has implications for endoscopic surveillance of individuals at risk.

Hereditary diffuse gastric cancer (HDGC) is caused by germline E-cadherin (CDH1) mutations in 25-40% of tested families. Management options for asymptomatic mutation carriers are fraught, since endoscopic surveillance can miss cancer foci and prophylactic gastrectomy has profound clinical sequelae. The aims of this study were to evaluate the impact of current surveillance practices on pre-operative diagnosis and to characterize the microscopic lesions in gastrectomy specimens to better inform clinical practice. Histological assessment and mapping of endoscopic surveillance and gastrectomy specimens were performed for eight asymptomatic CDH1 mutation carriers. E-cadherin expression and proliferation were analysed and evidence of epithelial-mesenchymal transition (EMT) was sought by immunohistochemistry for vimentin and cytokeratin 8/18. Four of eight patients had lesions detected at endoscopic surveillance. A median of 20.5 (range 0-66) signet ring foci were identified per gastrectomy (including in situ lesions and pagetoid spread). Foci were predominantly identified in the fundus and body (90% endoscopic biopsies and 85% in gastrectomy). The likelihood of detecting foci pre-operatively was positively correlated with the number of biopsies taken and the number of lesions in the gastrectomy specimen. E-cadherin expression in gastrectomy specimens was reduced or absent in all of the foci compared with the intervening gastric tissue, suggesting that these lesions are polyclonal. The foci had a low proliferative index (<2%) and there was no evidence for EMT. Multiple endoscopic biopsy sampling of the gastric mucosa increases the yield of microscopic cancer foci. The low proliferative index and lack of EMT suggests that these foci may represent an indolent stage of HDGC.

Mechanisms and sequelae of E-cadherin silencing in hereditary diffuse gastric cancer.

Around 25-40% of cases of hereditary diffuse gastric cancer (HDGC) are caused by heterozygous E-cadherin (CDH1) germline mutations. The mechanisms for loss of the second allele still remain unclear. The aims of this study were to elucidate mechanisms for somatic inactivation of the wild-type CDH1 allele and to seek evidence for cadherin switching. Archival tumour material was analysed from 16 patients with CDH1 germline mutations and seven patients fulfilling HDGC criteria without CDH1 germline mutations. The 16 CDH1 exons were sequenced. E-cadherin promoter methylation was analysed by bisulphite sequencing and pyrosequencing and allele specificity was determined using polymorphic loci. Loss of heterozygosity was analysed using microsatellite markers. Cadherin expression levels were determined by real-time RT-PCR and immunohistochemistry. Six of 16 individuals with germline mutations had at least one second hit mechanism. Two exonic mutations (exon 9 truncating, exon 3 missense) and four intronic mutations which may affect splicing were identified. Tumours from 4/16 individuals had promoter hypermethylation that was restricted to the A allele haplotype in three cases. E-cadherin loss (mRNA and protein) generally correlated with identification of a second hit. In cases without germline E-cadherin mutations there was no evidence for somatic mutation or significant promoter methylation. P-cadherin (>25% cells) was expressed in 7/13 (54%) and 4/5 (80%) with and without germline CDH1 mutations, respectively, independent of complete E-cadherin loss. Overall, inactivation of the second CDH1 allele occurs by mutation and methylation events. Methylation is commonly allele-specific and is uncommon without germline mutations. P-cadherin over-expression commonly occurs in individuals with diffuse type gastric cancer.

A novel role for the retinoic acid-catabolizing enzyme CYP26A1 in Barrett's associated adenocarcinoma.

Vitamin A deficiency is associated with carcinogenesis, and upregulation of CYP26A1, a major retinoic acid (RA)-catabolizing enzyme, has recently been shown in cancer. We have previously demonstrated alterations of RA biosynthesis in Barrett's oesophagus, the precursor lesion to oesophageal adenocarcinoma. The aims of this study were to determine CYP26A1 expression levels and functional effects in Barrett's associated carcinogenesis. Retinoic acid response element reporter cells were used to determine RA levels in non-dysplastic and dysplastic Barrett's cell lines and endoscopic biopsies. CYP26A1 expression levels, with or without induction by RA and lithocholic acid, were determined by quantitative reverse transcriptase-PCR (RT-PCR) and immunohistochemistry. CYP26A1 promoter activity was determined by a luciferase reporter construct. CYP26A1 was stably overexpressed in GihTERT cells, which were evaluated for gene-expression changes (pathway array and quantitative RT-PCR), cellular proliferation (cytometric DNA profile and colorimetric assay) and invasion (in vitro matrigel assay) with or without the CYP inhibitor ketaconazole. RA levels decreased progressively with the degree of dysplasia (P<0.05) and were inversely correlated with CYP26A1 gene levels and activity (P<0.01). CYP26A1 expression was increased synergistically by RA and lithocholic acid (P<0.05). Overexpression of CYP26A1 led to induction of c-Myc, epidermal growth factor receptor and matrix metalloproteinase 3 as well as downregulation of tissue inhibitor metalloproteinase 1 and 3. Functional effects of CYP26A1 overexpression were increased proliferation (P<0.01) and invasion in vitro (P<0.01), which were inhibited by ketaconazole. Overexpression of CYP26A1 causes intracellular RA depletion and drives the cell into a highly proliferative and invasive state with induction of other known oncogenes.

Impaired transforming growth factor beta signalling in Barrett's carcinogenesis due to frequent SMAD4 inactivation.

BACKGROUND AND AIMS: Transforming growth factor beta (TGF-beta) is frequently involved in gastrointestinal carcinogenesis although its contribution to oesophageal adenocarcinoma (AC) and its precursor Barrett's oesophageal epithelium (BE) metaplasia are unclear. METHODS: Expression of TGF-beta signalling components was assessed by reverse transcription-polymerase chain reaction (PCR), western blot, and immunohistochemistry in oesophageal endoscopic biopsies and cell lines. Genomic alterations in SMAD4 were characterised by fluorescence in situ hybridisation, methylation specific PCR, and sequencing. Functional integrity of TGF-beta signalling was assessed by characterisation of p21 and proliferation status. Smad4 negative BIC-1 cells were transiently transfected with smad4 and TGF-beta responsiveness evaluated. RESULTS: smad4 mRNA expression was progressively reduced in the metaplasia-dysplasia-adenocarcinoma sequence (p<0.01). A quarter of AC samples displayed an abnormal Smad4 protein isoform, with no corresponding changes in gene sequence or organisation. Methylation of smad4 has not been described previously but we found promoter methylation in 70% of primary AC samples. In 6/8 oesophageal cell lines, chromosomal rearrangements affected the smad4 locus. Lack of smad4 expression in BIC-1 cells occurred secondary to loss of one copy and extensive deletion of the second allele's promoter region. TGF-beta dependent induction of p21 and downregulation of minichromosome maintenance protein 2 was lost in >80% of BE and AC. TGF-beta failed to inhibit proliferation in 5/8 oesophageal cell lines. In BIC-1, the antiproliferative response was restored following transient transfection of smad4 cDNA. CONCLUSIONS: In BE carcinogenesis, downregulation of Smad4 occurs due to several different mechanisms, including methylation, deletion, and protein modification. Frequent alterations in TGF-beta signalling lead to a functionally significant impairment of TGF-beta mediated growth suppression.

Inflammatory gradient in Barrett's oesophagus: implications for disease complications.

INTRODUCTION: Barrett's oesophageal epithelium (BE) is clinically important due to the associated inflammatory and malignant complications which are unevenly distributed throughout the BE segment. As the immunoregulatory environment may influence disease manifestations, we analysed the inflammatory and cytokine responses throughout the BE mucosa. We then investigated whether the inflammatory gradient is related to the distribution of metaplastic cell subtypes, epithelial exposure to the components of refluxate, or squamocolumnar cell interactions. METHODS: Fifty consecutive patients with long segment BE were recruited. The segmental degree of endoscopic and histopathological inflammation was graded, and expression of interleukin (IL)-1 beta, IL-8, IL-4, and IL-10 were determined by ELISA following organ culture with or without addition of acid or bile salts. Mucin staining and IL-10 immunohistochemistry were performed. The effect of squamocolumnar interactions on cytokine expression were analysed using cocultures of squamous (OE-21) and BE (TE7) carcinoma cell lines. RESULTS: There was a histopathological inflammatory gradient in BE. Inflammation was maximal at the new squamocolumnar junction with > or = 2-fold increase in proinflammatory IL-8 and IL-1 beta expression. The proximal proinflammatory response could not be explained by the distribution of metaplastic subtypes. Pulsatile exposure of BE to acid and bile, as well as juxtaposition of BE to squamous epithelial cells in culture, increased expression of IL-1 beta. In contrast, inflammation was minimal distally with a significant increase in anti-inflammatory IL-10 expression and 4/6 cancers occurred distally. CONCLUSIONS: Specific cytokine responses may contribute to the localisation of inflammatory and malignant complications within BE.