Abstract 3991: Standardization and optimization of circulating microRNA serum profiling in patients with cancer
Authors: Poel Dennis , Voortman Johannes , van den Oord Rosanne , Gall Helen , Verheul Henk M.W. 
Published: August 2015

Background Circulating microRNAs (miRNAs) in blood have recently emerged as promising non-invasive biomarkers for cancer diagnosis and prognosis. Standardization of circulating miRNA profiling methodology is lacking. This may explain contradictory results reported in the same tumor type.

We performed a comparative analysis of different extraction methods for circulating miRNA profiling in patients with cancer. Furthermore, several pre-analytical factors that might confound miRNA recovery were assessed to determine their influence on the serum miRNA profile.

Methods Commercially available RNA isolation kits were compared by performing targeted miRNA expression profiling in pooled serum from patients with cancer and healthy donors. MiRNA expression was determined by qRT-PCR using Taqman microRNA Assays or Exiqon ExiLENT SYBR Green. Normalization was performed using miR-16-5p and an RNA spike (cel-miR-39) for quality control. For each RNA isolation kit, the effect of RNA carriers glycogen, bacteriophage MS2 or their combination was assessed.

To establish the temporal fluctuation of miRNA expression in individual patients we designed a pilot study in a cohort of 10 head and neck cancer patients. We compared the circulating miRNA profile of two pretreatment serum samples which were collected with a minimum interval of 24 hours.

The influence of serum coagulation time and serum indices for: hemolysis, lipemia and icterus (Roche Modular) on the miRNA profile was assessed.

Results The use of the miRCURY kit (Exiqon), measuring miR-16, miR-16-5p and miR-20a, resulted in the most reproducible miRNA serum profile. The addition of glycogen promoted the miRNA extraction yield (average Ct value decreased from 27.43 to 26.26), but resulted in decreased reproducibility (standard error of mean (SEM) 0.40 vs 0.92, respectively). Reproducibility of miRNA detection could be restored after the addition of MS2. We demonstrate that combining RNA carriers MS2 and glycogen with the miRCURY isolation kit results in enhanced reproducibility and RNA yield (avg. raw Ct 25.94±SEM 0.30). Targeted detection of 5 miRNAs in serum from 9 patients with cancer and 10 healthy donors demonstrated high reproducibility of miRNA levels in both groups (SEM range: 0.16 to 0.49). Standardization of the method is ongoing.

Conclusion Circulating miRNAs are emerging as potential prognostic and predictive biomarkers in cancer. We here demonstrate a non-invasive, optimized method for miRNA profiling from clinical blood samples. Intra,- and interexperimental miRNA expression levels were highly reproducible. Developing a standardized, optimized method for miRNA profiling is pivotal for future clinical applications.

Citation Format: Dennis Poel, Johannes Voortman, Rosanne van den Oord, Helen Gall, Henk M.W. Verheul. Standardization and optimization of circulating microRNA serum profiling in patients with cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3991. doi:10.1158/1538-7445.AM2015-3991