Whole-miRNome profiling identifies prognostic serum miRNAs in esophageal adenocarcinoma: the influence of Helicobacter pylori infection status

Abstract

Cell free circulating microRNAs (cfmiRNAs) have been recognized as robust and stable biomarkers of cancers. However, little is known about the prognostic significance of cfmiRNAs in esophageal adenocarcinoma (EA). In this study, we explored whether specific cfmiRNA profiles could predict EA prognosis and whether Helicobacter pylori (HP) infection status could influence the association between cfmiRNAs and EA survival outcome. We profiled 1075 miRNAs in pooled serum samples from 30 EA patients and 30 healthy controls. The most relevant cfmiRNAs were then assessed for their associations with EA survival in an independent cohort of 82 patients, using Log-rank test and multivariate Cox regression models. Quantitative real-time PCR (qRT-PCR) was used for cfmiRNA profiling. HP infection status was determined by immunoblotting assay. We identified a panel of 18 cfmiRNAs that could distinguish EA patients from healthy subjects (P = 3.0E-12). In overall analysis and in HP-positive subtype patients, no cfmiRNA was significantly associated with EA prognosis. In HP-negative patients, however, 15 cfmiRNAs were significantly associated with overall survival (OS) (all P < 0.05). A combined 2-cfmiRNA (low miR-3935 and high miR-4286) risk score was constructed; that showed greater risk for worse OS (HR = 2.22, P = 0.0019) than individual cfmiRNA alone. Patients with high-risk score had >10-fold increased risk of death than patients with low risk score (P = 0.0302; HR = 10.91; P = 0.0094). Our findings suggest that dysregulated cfmiRNAs may contribute to EA survival outcome and HP infection status may modify the association between cfmiRNAs and EA survival.

© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Figures

Fig. 1.

Study flow chart.

Fig. 2.

Volcano plot illustrates the frequency distribution of FC values and the distribution of FC with corresponding P-values (all values were log transformed). The vertical dash lines represent the cut-off levels of upper 95th percentile and lower 5th percentile of FC values, respectively. The horizontal dash line indicates the significant level (0.05) in log10 scale. cfmiRNAs with FCs either above the 95th percentile or below the 5th percentile and with P-values <0.05 were selected as the most differentiated cfmiRNAs. FC, fold change.

Fig. 3.

Hierarchical cluster analysis demonstrates that a panel of 18 cfmiRNAs could clearly distinguish EA cases from controls (P = 3.0E–12, Hotelling T test).

Fig. 4.

Survival analysis of the cfmiRNA score. (A) Kaplan–Meier estimates of the cfmiRNA score identified by Cox regression model. Patients were classified into low risk group (reference) and high risk group by median stratification. (B) Cox regression analysis of the cfmiRNA risk score in HP-negative patients, adjusting for age, sex, stages, performance status, smoking sort, body mass index at diagnosis and GERD symptoms.