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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.

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.

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.

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.

Many patients with Oesophageal Adenocarcinoma (OAC) do not benefit from chemoradiotherapy treatment due to therapy resistance. To better understand the mechanisms involved in resistance and to find potential biomarkers, we investigated the association of microRNAs, which regulate gene expression, with the response to individual treatments, focusing on radiation. Intrinsic radiation resistance and chemotherapy drug resistance were assessed in eight OAC cell lines, and miRNA expression profiling was performed via TaqMan OpenArray qPCR. miRNAs discovered were either uniquely associated with resistance to radiation, cisplatin, or 5-FU, or were common to two or all three of the treatments. Target mRNA pathway analyses indicated several potential mechanisms of treatment resistance. miRNAs associated with the in vitro treatment responses were then investigated for association with pathologic response to neoadjuvant chemoradiotherapy (nCRT) in pre-treatment serums of patients with OAC. miR-451a was associated uniquely with resistance to radiation treatment in the cell lines, and with the response to nCRT in patient serums. Inhibition of miR-451a in the radiation resistant OAC cell line OE19 increased radiosensitivity (Survival Fraction 73% vs. 87%, p = 0.0003), and altered RNA expression. Pathway analysis of effected small non-coding RNAs and corresponding mRNA targets suggest potential mechanisms of radiation resistance in OAC.

Introduction Outcome following fundoplication for gastroesophageal reflux can be measured using objective tests, symptom scores and quality of life (QoL) measures. Which is best and how these assessments correlate is uncertain. To determine the utility of assessment measures we compared a general QoL measure (SF-36) and a disease-specific measure (GERD-hr-QoL) with symptom and satisfaction scores in individuals following fundoplication. Methods 329 individuals underwent fundoplication between 2000 and 2015 in 2 centres in Australia and the Netherlands. Patients were assessed before and 3, 12 and 24 months after surgery using 10-point Likert scales to assess heartburn and satisfaction, the SF-36 questionnaire and the GERD-hr-QoL questionnaire. SF-36 scores were converted into component scores: Physical Component Scale (PCS) score and Mental Component Scale (MCS) score. Correlations between QoL measures and clinical outcomes were determined. Results Surgery relieved heartburn (7.0 vs. 0.0 median, P  < 0.001) and patients were highly satisfied with the outcome (median 9.0). PCS and MCS scores improved after surgery (PCS 40.9 vs. 46.0, P  < 0.001; MCS 47.6 vs. 50.3, P  = 0.027). GERD-hr-QoL scores also improved after surgery (15.7 vs. 3.7, P  < 0.001). Correlations between PCS and MCS scores versus heartburn and satisfaction scores were generally weak or absent. However, correlations between GERD-hr-QoL versus heartburn and satisfaction scores were moderate to strong. Conclusion Despite improvements in scores, the SF-36 correlated poorly with clinical outcome measures, and its use to measure outcome following fundoplication is questioned. However, the GERD-hr-QoL correlated well with the symptom scores, suggesting this disease-specific QoL measure is a better tool for assessing anti-reflux surgery outcome.

BackgroundClinical trials report improved overall survival following neoadjuvant chemoradiotherapy in patients undergoing surgery for esophageal adenocarcinoma, with a 10–15% survival improvement. MicroRNAs (miRNAs) are small noncoding RNAs that are known to direct the behavior of cancers, including response to treatment. We investigated the ability of miRNAs to predict outcomes after neoadjuvant chemoradiotherapy.MethodsEndoscopic biopsies from esophageal adenocarcinomas were obtained before neoadjuvant chemoradiotherapy and esophagectomy. miRNA levels were measured in the biopsies using next generation sequencing and compared with pathological response in the surgical resection, and subsequent survival. miRNA ratios that predicted pathological response were identified by Lasso regression and leave-one-out cross-validation. Association between miRNA ratio candidates and relapse-free survival was assessed using Kaplan–Meier analysis. Cox regression and Harrell’s C analyses were performed to assess the predictive performance of the miRNAs.ResultsTwo miRNA ratios (miR-4521/miR-340-5p and miR-101-3p/miR-451a) that predicted the pathological response to neoadjuvant chemoradiotherapy were found to be associated with relapse-free survival. Pretreatment expression of these two miRNA ratios, pretreatment tumor differentiation, posttreatment AJCC histopathological tumor regression grading, and posttreatment tumor clearance/margins were significant factors associated with survival in Cox regression analysis. Multivariate analysis of the two ratios together with pretherapy factors resulted in a risk prediction accuracy of 85% (Harrell’s C), which was comparable with the prediction accuracy of the AJCC treatment response grading (77%).ConclusionsmiRNA-ratio biomarkers identified using next generation sequencing can be used to predict disease free survival following neoadjuvant chemoradiotherapy and esophagectomy in patients with esophageal adenocarcinoma.

Background The polymerase chain reaction amplifies and quantifies small amounts of DNA. It is a cyclic process, during each cycle of which each strand of template DNA is copied with probability approaching one: the amount of DNA approximately doubles and this amount can be estimated fluorimetrically each cycle, producing a set of fluorescence values hereafter referred to as the amplification curve. Commonly the biological question of relevance is one of the ratio of DNA concentrations in two samples: a ratio that is deduced by comparing the two amplification curves, usually by way of a plot of fluorescence against cycle number. Central to this analysis is measuring the extent to which one amplification curve is shifted relative to the other, a measurement often accomplished by defining a threshold or quantification cycle, C q , for each curve: the fractional cycle number at which fluorescence reaches some threshold or at which some other criterion (maximum slope, maximum rate of change of slope) is satisfied. We propose an alternative where position is measured relative to a reference curve; position equates to the cycle shift which maximizes the correlation between the reference and the observed fluorescence sequence. A key parameter of the reference curve is obtained by fixed-point convergence. Results We consider the analysis of dilution series constructed for the estimation of qPCR amplification efficiency. The estimate of amplification efficiency is based on the slope of the regression line when the C q is plotted against the logarithm of dilution. We compare the approach to three commonly used methods for determining C q ; each is applied to publicly accessible calibration data sets, and to ten from our own laboratory. As in the established literature we judge their relative merits both from the standard deviation of the slope of the calibration curve, and from the variance in C q for replicate fluorescence curves. Conclusions The approach does not require modification of experimental protocols, and can be applied retrospectively to existing data. We recommend that it be added to the methodological toolkit with which laboratories interpret their real-time PCR data.

Background Endoscopic surveillance of Barrett’s esophagus (BE) is probably not cost-effective. A sub-population with BE at increased risk of high-grade dysplasia (HGD) or esophageal adenocarcinoma (EAC) who could be targeted for cost-effective surveillance was sought. Methods The outcome for BE surveillance from 2003 to 2012 in a structured program was reviewed. Incidence rates and incidence rate ratios for developing HGD or EAC were calculated. Risk stratification identified individuals who could be considered for exclusion from surveillance. A health-state transition Markov cohort model evaluated the cost-effectiveness of focusing on higher-risk individuals. Results During 2067 person-years of follow-up of 640 patients, 17 individuals progressed to HGD or EAC (annual IR 0.8%). Individuals with columnar-lined esophagus (CLE) ≥2 cm had an annual IR of 1.2% and >8-fold increased relative risk of HGD or EAC, compared to CLE <2 cm [IR—0.14% (IRR 8.6, 95% CIs 4.5–12.8)]. Limiting the surveillance cohort after the first endoscopy to individuals with CLE ≥2 cm, or dysplasia, followed by a further restriction after the second endoscopy—exclusion of patients without intestinal metaplasia—removed 296 (46%) patients, and 767 (37%) person-years from surveillance. Limiting surveillance to the remaining individuals reduced the incremental cost-effectiveness ratio from US$60,858 to US$33,807 per quality-adjusted life year (QALY). Further restrictions were tested but failed to improve cost-effectiveness. Conclusions Based on stratification of risk, the number of patients requiring surveillance can be reduced by at least a third. At a willingness-to-pay threshold of US$50,000 per QALY, surveillance of higher-risk individuals becomes cost-effective.