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Background The poor prognosis and rising incidence of esophageal adenocarcinoma highlight the need for improved detection methods. The potential for circulating microRNAs (miRNAs) as biomarkers in other cancers has been shown, but circulating miRNAs have not been well characterized in esophageal adenocarcinoma. We investigated whether circulating exosomal miRNAs have potential to discriminate individuals with esophageal adenocarcinoma from healthy controls and non-dysplastic Barrett’s esophagus. Methods Seven hundred fifty-eight miRNAs were profiled in serum circulating exosomes from a cohort of 19 healthy controls, 10 individuals with Barrett’s esophagus, and 18 individuals with locally advanced esophageal adenocarcinoma. MiRNA expression was assessed using all possible permutations of miRNA ratios per individual. Four hundred eight miRNA ratios were differentially expressed in individuals with cancer compared to controls and Barrett’s esophagus (Mann-Whitney U test, P  < 0.05). The 179/408 ratios discriminated esophageal adenocarcinoma from healthy controls and Barrett’s esophagus (linear regression, P  < 0.05; area under receiver operating characteristic (ROC) > 0.7, P  < 0.05). A multi-biomarker panel (RNU6-1/miR-16-5p, miR-25-3p/miR-320a, let-7e-5p/miR-15b-5p, miR-30a-5p/miR-324-5p, miR-17-5p/miR-194-5p) demonstrated enhanced specificity and sensitivity (area under ROC = 0.99, 95 % CI 0.96–1.0) over single miRNA ratios to distinguish esophageal adenocarcinoma from controls and Barrett’s esophagus. Conclusions This study highlights the potential for serum exosomal miRNAs as biomarkers for the detection of esophageal adenocarcinoma.

Loss of A, B and H antigens from the red blood cells of patients with myeloid malignancies is a frequent occurrence. Previously, we have reported alterations in ABH antigens on the red blood cells of 55% of patients with myeloid malignancies. To determine the underlying molecular mechanisms of this loss, we assessed ABO allelic expression in 21 patients with ABH antigen loss previously identified by flow cytometric analysis as well as an additional 7 patients detected with ABH antigen changes by serology. When assessing ABO mRNA allelic expression, 6/12 (50%) patients with ABH antigen loss detected by flow cytometry and 5/7 (71%) of the patients with ABH antigen loss detected by serology had a corresponding ABO mRNA allelic loss of expression. We examined the ABO locus for copy number and DNA methylation alterations in 21 patients, 11 with loss of expression of one or both ABO alleles, and 10 patients with no detectable allelic loss of ABO mRNA expression. No loss of heterozygosity (LOH) at the ABO locus was observed in these patients. However in 8/11 (73%) patients with loss of ABO allelic expression, the ABO promoter was methylated compared with 2/10 (20%) of patients with no ABO allelic expression loss (P = 0.03). We have found that loss of ABH antigens in patients with hematological malignancies is associated with a corresponding loss of ABO allelic expression in a significant proportion of patients. Loss of ABO allelic expression was strongly associated with DNA methylation of the ABO promoter.

Background Ulceration of the oesophageal squamous mucosa (ulcerative oesophagitis) is a pathological manifestation of gastro-oesophageal reflux disease, and is a major risk factor for the development of Barrett’s oesophagus. Barrett’s oesophagus is characterised by replacement of reflux-damaged oesophageal squamous epithelium with a columnar intestinal-like epithelium. We previously reported discovery of microRNAs that are differentially expressed between oesophageal squamous mucosa and Barrett’s oesophagus mucosa. Now, to better understand early steps in the initiation of Barrett’s oesophagus, we assessed the expression, location and function of these microRNAs in oesophageal squamous mucosa from individuals with ulcerative oesophagitis. Methods Quantitative real-time PCR was used to compare miR-21, 143, 145, 194, 203, 205 and 215 expression levels in oesophageal mucosa from individuals without pathological gastro-oesophageal reflux to individuals with ulcerative oesophagitis. Correlations between microRNA expression and messenger RNA differentiation markers BMP-4, CK8 and CK14 were analyzed. The cellular localisation of microRNAs within the oesophageal mucosa was determined using in-situ hybridisation. microRNA involvement in proliferation and apoptosis was assessed following transfection of a human squamous oesophageal mucosal cell line (Het-1A). Results miR-143, miR-145 and miR-205 levels were significantly higher in gastro-oesophageal reflux compared with controls. Elevated miR-143 expression correlated with BMP-4 and CK8 expression, and elevated miR-205 expression correlated negatively with CK14 expression. Endogenous miR-143, miR-145 and miR-205 expression was localised to the basal layer of the oesophageal epithelium. Transfection of miR-143, 145 and 205 mimics into Het-1A cells resulted in increased apoptosis and decreased proliferation. Conclusions Elevated miR-143, miR-145 and miR-205 expression was observed in oesophageal squamous mucosa of individuals with ulcerative oesophagitis. These miRNAs localised to the basal layer of the oesophageal epithelium. They reduced proliferation and increased apoptosis, and may play roles in regulating epithelial restoration in response to injury caused by gastro-oesophageal reflux.

TP53 gene mutations occur in 70% of oesophageal adenocarcinomas (OACs). Given the central role of p53 in controlling cellular response to therapy we investigated the role of mutant (mut-) p53 and SLC7A11 in a CRISPR-mediated JH-EsoAd1 TP53 knockout model. Response to 2 Gy irradiation, cisplatin, 5-FU, 4-hydroxytamoxifen, and endoxifen was assessed, followed by a TaqMan OpenArray qPCR screening for differences in miRNA expression. Knockout of mut-p53 resulted in increased chemo- and radioresistance (2 Gy survival fraction: 38% vs. 56%, p < 0.0001) and in altered miRNA expression levels. Target mRNA pathways analyses indicated several potential mechanisms of treatment resistance. SLC7A11 knockdown restored radiosensitivity (2 Gy SF: 46% vs. 73%; p = 0.0239), possibly via enhanced sensitivity to oxidative stress. Pathway analysis of the mRNA targets of differentially expressed miRNAs indicated potential involvement in several pathways associated with apoptosis, ribosomes, and p53 signaling pathways. The data suggest that mut-p53 in JH-EsoAd1, despite being classified as non-functional, has some function related to radio- and chemoresistance. The results also highlight the important role of SLC7A11 in cancer metabolism and redox balance and the influence of p53 on these processes. Inhibition of the SLC7A11-glutathione axis may represent a promising approach to overcome resistance associated with mut-p53.

Background Neoadjuvant treatment plays a crucial role in the therapy of advanced esophageal cancer. However, response to radiochemotherapy varies widely. Proton pump inhibitors (PPIs) have been demonstrated to impact on chemotherapy in a variety of other cancers. We analyzed the impact of PPI treatment on esophageal cancer cell lines, and investigated mechanisms that mediate the effect of PPI treatment in this tumour. Methods We investigated the effect of esomeprazole treatment on cancer cell survival, adhesion, migration and chemotherapy in human adeno-(OE19) and squamous-cell-carcinoma (KYSE410) cell lines. Furthermore, we investigated the effect of PPI treatment on intra-/extracellular pH and on expression of resistance-relevant miRNAs. Results Esomeprazole significantly inhibited tumour cell survival (in a dose-dependent manner), adhesion and migration in both tumour subtypes. Furthermore, esomeprazole augmented the cytotoxic effect of cisplatin and 5-FU in both tumour subtypes. Surprisingly, PPI treatment led to a significant increase of intracellular pH and a decrease of the extracellular pH. Finally, we found esomeprazole affected expression of resistance-relevant miRNAs. Specifically, miR-141 and miR-200b were upregulated, whereas miR-376a was downregulated after PPI treatment in both tumour types. Conclusion Our study demonstrates for the first time that PPIs impact on tumour cell survival, metastatic potential and sensitivity towards chemotherapy in esophageal cancer cell lines. Furthermore, we observed that in this tumour entity, PPIs do not lead to intracellular acidification, but affect the expression of resistance-relevant miRNAs.

Background Response to chemotherapy varies widely in patients with advanced oesophageal cancer. We investigated the impact of manipulating certain microRNAs on response to cisplatin and 5-fluorouracil (5-FU) in oesophageal cancer cells. Methods Cisplatin-/5-fluorouracil-resistant oesophageal squamous cell carcinoma (SCC) and adenocarcinoma (EAC) cell lines were established, and the impact of ectopic upregulation of miR-106a and miR-148a on response to both drugs was assessed. Results The impact of miR-106a-upregulation was inconsistent. Upregulation was followed by reduced sensitivity to cisplatin in chemotherapy-sensitive EAC cells (cell survival, +8.7 ± 0.8%; p = 0.003) and an improved response to 5-FU in cisplatin-resistant EAC cells (cell survival, −6.4 ± 2.5%; p = 0.011). MiR-148a upregulation significantly increased sensitivity to chemotherapy in seven out of ten cell lines, represented by a decrease in cell viability of 22.6 ± 7.9% to 50.5 ± 10.6% after cisplatin ( p ≤ 0.014) and 6.0 ± 0.8% to 15.0 ± 4.1% after 5-FU treatment ( p ≤ 0.012). The only cell lines in which miR-148a upregulation had no effect were cisplatin-resistant EAC exposed to cisplatin and 5-FU-sensitive and 5-FU-resistant SCC cells exposed to 5-FU. Conclusion MiR-148a sensitized chemotherapy-sensitive oesophageal cancer cell lines to cisplatin and, to a lesser extent, to 5-flurouracil and attenuated resistance in chemotherapy-resistant variants. Further experimental and clinical studies to investigate the exact mechanisms involved are warranted.

The biomarker development field within molecular medicine remains limited by the methods that are available for building predictive models. We developed an efficient method for conservatively estimating confidence intervals for the cross validation-derived prediction errors of biomarker models. This new method was investigated for its ability to improve the capacity of our previously developed method, StaVarSel, for selecting stable biomarkers. Compared with the standard cross validation method, StaVarSel markedly improved the estimated generalisable predictive capacity of serum miRNA biomarkers for the detection of disease states that are at increased risk of progressing to oesophageal adenocarcinoma. The incorporation of our new method for conservatively estimating confidence intervals into StaVarSel resulted in the selection of less complex models with increased stability and improved or similar predictive capacities. The methods developed in this study have the potential to improve progress from biomarker discovery to biomarker driven translational research.

Introduction Ablation of Barrett’s esophagus using Argon plasma coagulation (APC) is usually followed by the formation of a neosquamous epithelium. Investigating simple columnar or stratified squamous epithelium associated cytokeratin and microRNA (miRNA) expression in neo-squamous epithelium could help determine the identity and stability of the neosquamous epithelium. Methods Nine patients underwent ablation of Barrett’s esophagus with APC. Biopsies were collected from Barrett’s esophagus mucosa and proximal normal squamous epithelium before ablation, and from neosquamous and normal squamous epithelium after ablation. Additional esophageal mucosal biopsies from ten nonrefluxing subjects were used as a reference. RNA was extracted and real-time polymerase chain reaction was used to measure the expression of the cytokeratins CK-8 and CK-14 and the microRNAs miR-143 and miR-205. Results CK-8 and miR-143 expression were significantly higher in Barrett’s esophagus mucosa, compared to neosquamous and normal squamous epithelium before and after APC, whereas miRNA-205 and CK-14 expression was significantly lower in Barrett’s esophagus mucosa compared to all categories of squamous mucosa. The expression of CK-8, CK-14, miR-205, and miR-143 was similar between neosquamous epithelium compared to normal squamous epithelium in patients with Barrett’s esophagus. Only miR-143 expression was significantly higher in neosquamous and normal squamous epithelium before and after APC compared to normal squamous epithelium from control subjects ( p  < 0.004). Conclusions The expression levels of cytokeratins and miRNAs studied in post-ablation neosquamous epithelium and normal squamous epithelium in patients with Barrett’s esophagus are similar. In patients with Barrett’s esophagus, miR-143 expression is still elevated in both neosquamous mucosa, and the squamous mucosa above the metaplastic segment, suggesting that this mucosa may not be normal; i.e., it is different to that seen in subjects without Barrett’s esophagus. miR-143 could promote a Barrett’s epithelium gene expression pattern, and this could have a role in development of Barrett’s esophagus.

Resistance to radiation is considered to be an important reason for local failure after radiotherapy and tumor recurrence. However, the exact mechanisms of tumor resistance remain poorly understood. Current investigations of microRNAs as potential diagnostic and therapeutic tools for cancer treatment have shown promising results. With respect to radiotherapy resistance and response, there is now emerging evidence that microRNAs modulate key cellular pathways that mediate response to radiation. These data suggest that microRNAs might have significant potential as targets for the development of new therapeutic strategies to overcome radioresistance in cancer. This review summarizes the current literature pertinent to the influence of microRNAs in the response to radiotherapy for cancer treatment, with an emphasis on microRNAs as novel diagnostic and prognostic markers, as well as their potential to alter radiosensitivity.