Abstract 1793: Optimal quantification of a circulating miRNA profile predictive for treatment response with droplet digital PCR in patients with multi-organ metastatic colorectal cancer
Authors: Bakkerus Lotte , Poel Dennis , Gootjes Elske C. , Verhoef Cornelis , Buffart Tineke E. , Verheul Hendrik M. 
Published: August 2019

Abstract
Introduction: Presence of microRNAs (miRNAs) in blood specimens hold promise for a predictive biomarker for treatment response in clinical practice as they are relatively resistant to degradation. Previously, we identified a six miRNA expression signature from tumor tissue that predicted response to first line systemic treatment in patients with metastatic colorectal cancer (mCRC) when combined with clinicopathological factors. The six miRNA expression signature consists of miR-17-5p, miR-20a-5p, miR-30a-5p, miR-92a-3p, miR-92b-3p and miR-98-5p. The aim of this study is to determine whether circulating levels of these miRNAs will be predictive for treatment outcome as well. Therefore we first studied whether these six miRNAs can be adequately measured in blood samples of patients with mCRC undergoing first line chemotherapy. Droplet digital PCR (ddPCR) technology was used for optimal quantification of miRNAs compared to normal quantitative PCR, because of low abundance of tumor specific miRNAs in blood.
Methods: Serum samples of 120 patients with multi-organ mCRC who participated in a multi-center randomized controlled clinical trial were analyzed. RNA was isolated with miRNeasy Serum/Plasma Advanced Kit of Qiagen. Expression levels of the six miRNA expression signature was determined with ddPCR by using the miRCURY LNA miRNA PCR system combined with EvaGreen. Primer concentration and annealing temperature were first optimized per specific miRNA assay for ddPCR. RNA spike cel-miR-39-3p was used for quality control. Analysis was performed on samples with at least a minimal amount of 10.000 obtained droplets. Absolute quantification per patient is displayed as copies per µl (cop/µl) as defined by Quantasoft software.
Results: In 120 blood samples miRNAs were measured by ddPCR. The most optimal primer concentrations for the different miRNAs was 0,5 µl for cel-miR-39-3p and 1,0 µl for the other 6 miRNAs. For cel-miR-39-3p, miR-17-5p, miR-20a-5p and miR-92a-3p the most efficient annealing temperature was 53 °C and for miR-30a-5p, miR-92b-3p and miR-98-5p this was 58 °C. The mean number of obtained droplets was 15.044 (SD 1922). The highest amount of detected copies were observed in miR-17-5p (644,4 cop/µl), miR-20a-5p (1037,1 cop/µl) and miR-92a-3p (1136,0 cop/µl). Lower expression levels were observed in miR-30a-5p (9,88 cop/µl), miR-92b-3p (1,6 cop/µl) and miR-98-5p (6,9 cop/µl).
Conclusion: We demonstrated that with ddPCR absolute quantification of these six miRNAs in serum samples was efficient and reliable. Further analysis will reveal whether circulating miRNA expression levels can be used as a liquid biomarker for response to first line systemic treatment in patients with multi-organ mCRC.
Citation Format: Lotte Bakkerus, Dennis Poel, Elske C. Gootjes, Cornelis Verhoef, Tineke E. Buffart, Hendrik M. Verheul. Optimal quantification of a circulating miRNA profile predictive for treatment response with droplet digital PCR in patients with multi-organ metastatic colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1793.