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Plasma microRNAs in oesophageal and gastric cancer915BIO-RAD Laboratories, Veenendaal, the Netherlands). The data were analyzed with Quantasoft software (BIO-RAD). The cut-off for the number of quantified droplets was set on at least 10.000. Expression is defined as miRNA copy per 1 %u00b5l input normalized to cel-miR-39-3p [38]. Continuous miRNA expression levels measured with ddPCR were converted to a dichotomous variable using the median expression as a threshold. All experiments were carried out in accordance with institutional guidelines and regulations.Statistical analysis Overall survival and progression-free survival differences between patients divided based on median ci-miRNA expression in the baseline samples were calculated by performing a Kaplan-Meier analysis and a log-rank test. Analysis between the paired baseline and follow-up samples was performed using the Wilcoxon matched-pairs-signed rank test. ci-miRNA expression was compared between patients with or without clinical benefit (CB) of the chemotherapy for the subgroups GC, EC and EAC or GC (miR-200c-3p) using the Mann-Whitney U test. Patients who did not start with chemotherapy were excluded from this analysis. The Mann-Whitney U test was used to compare the ci-miRNA expression in the samples with vitamin supplementation. The statistical analysis was performed using GRAPHPAD PRISM 8 Software.ResultsPatient samplesA total of 225 plasma samples were collected originating from 68 patients during the randomized phase two study. From 63 patients a baseline sample was available (14GC; 49EC). A total of 53 follow up samples were available of these 63 patients (12GC; 41EC). Heparinase treatment optimization and sample inclusionmiRNA detection in the heparinized patient samples by RT-PCR was not possible without heparinase treatment. To optimize the heparinase treatment different RNA volumes incubated with different heparinase quantities were tested. Cel-miR39-3p quantification in RNA isolated from EDTA plasma without heparinase was used as a positive control. The mean Cq value of cel-miR-39-3p in this positive