Rrlc-Ms/Ms-Based Metabolomics Of Esophageal Cancer

A global and targeted metabolomics study, using plasma and urine from esophageal carcinoma (ESCC) patients before and after chemoradiotherapy (CRT) and healthy controls, was originally carried out by RRLC-(±)ESI-MS/MS to determine global alterations in the metabolic profiles and find biomarkers potentially applicable to diagnosis and monitoring treatment effects.To explore more reliable potential biomarker candidates, an independent test set model was applied and the predictive ability of the established model was evaluated. Discriminating variables were selected according to variable importance in projection values (VIP), S-plot, jack-knifed-based confidence intervals, raw data plot, an independent t-test, R-package CAMERA, partial correlation analysis, and extracted ion chromatograms (XICs). Due to the cancers involve systematic deregulation of biochemical pathways, the single biomarker would be limited utilization for diagnosis. ROC analysis was conducted on single potential biomarkers and used for discovering more reliable biomarker panels. The exact mass, isotope pattern, the free databases search, high resolution MS/MS analyses were performed for the identification of the metabolites of interest. Together, biological significance of these metabolites was discussed. Understanding the biological significance of these potential biomarkers could provide further insight into the mechanisms underlying the pathophysiology of ESCC and treatment intervention, and facilitate the discovery of biomarkers for diagnosis, treatment monitoring, and response prediction in ESCC.For establishing the RRLC-MS method of the plasma metabolomics, the study was undertaken by comparing the different plasma preparation methods and the analytical condition in detail. The results indicated3-fold cold acetonitrile was optimal. Eventually, the method was evaluated by using ten representative compounds common in plasma. The results showed that the method was satisfactory and would be applied in plasma metabolomics. Then, a global metabolomics study using plasma from four groups including ESCC patients before, during and after CRT and healthy controls, was originally carried out by RRLC-MS to find biomarkers potentially applicable to diagnosis and monitoring treatment effects.52and48metabolites were found to be significantly altered in ESCC patients vs. healthy controls and in pre-vs. post-treatment patients based on the eatablished multivariate statistical data analysis (MVDA), respectively. Combined with the treatment effect,5metabolites were found to be closely related with the therapeutic response.In additon, to explore the potential biomarkers in urine samples, the sample preparation method was investigated. Creatinine-normalized dilution method was applied. Then, a global metabolomics using urine from ESCC patients (before and after CRT treatment) and healthy controls, was originally carried out by RRLC-MS.83and43metabolites were found to be significantly altered in ESCC patients vs. healthy controls and in pre-vs. post-treatment patients, respectively. Combined with the treatment effect, I metabolites were found to be closely related with the therapeutic response. Furthermore,5metabolites were picked out to be related with the disease staging.To further validate the reliability of the potential biomarkers, independent validation tests based on RRLC-MS/MS targeted metabolomics were performed for both urine and plasma samples. The resulted35and20metabolites were picked out after the validation test in comparision of ESCC patients vs. healthy controls and pre-vs. post-treatment patients in plasma.3metabolites were closely related with the treatment response. The resulted49and26metabolites were picked out after the validation test in comparision of ESCC patients vs. healthy controls and in pre-vs. post-treatment patients in urine.Finally,28metabolites were identified as diagnostic biomarkers, including18metabolites in plasma,15metabolites in urine,5of which were identified in both plasma and urine samples.16metabolites were identified as therapeutic biomarkers, including I1metabolites in plasma,7metabolites in urine,2of which were identified in both plasma and urine samples. In additon,4metabolites were identified as closely related with the treatment reaponse. Furthermore, according to the ROC analysis, two biomarker panels were generated with the high sensitivity and specificity for ESCC diagnosis.Together, the biological significance of these metabolites was discussed. Abnormal levels of these metabolites indicate that the purine metabolism, TCA cycle and β-oxidation, glycolysis metabolism are disturbed in ESCC patients. The result would be useful for the insight into occurrence and development of ESCC disease.