Evaluation of Storage Tubes for Combined Analysis of Circulating Nucleic Acids in Liquid Biopsies.

In the last decade, circulating nucleic acids such as microRNAs (miRNAs) and cell-free DNA (cfDNA) have become increasingly important in serving as potential novel biomarkers for a variety of human diseases. If cell-free nucleic acids are to become routinely used in diagnostics, the difference in plasma miRNA and cfDNA levels between healthy and diseased subjects must exceed pre-analytical and analytical variability. Until now, few studies have addressed the time limitations of pre-processing or explored the potential use of long-term blood storage tubes, which might need to be implemented in real-life diagnostics. In this study, we analyzed the stability of four breast cancer-associated miRNAs and two cancer-associated genes under various storage conditions, to test their limitations for potential application in clinical diagnostics. In two consecutive experiments, we tested the limits of conventional EDTA tubes, as well as long-term storage blood collection tubes (BCTs) from four different manufacturers. We found that circulating miRNAs are relatively stable when stored in EDTA monovettes for up to 12 h before processing. When stored in BCTs, circulating miRNAs and cfDNA are stable for up to 7 days, depending on the manufacturer. Norgen tubes were superior for cfDNA yield, while Streck tubes performed the worst in our study with hemolysis induction. In conclusion, plasma prepared from whole blood is suitable for the quantification of both cf-miRNAs and cfDNA simultaneously.

AAMP is a binding partner of costimulatory human B7-H3.

Background: Targeted immunotherapies are of growing interest in the treatment of various cancers. B7 homolog 3 protein (B7-H3), a member of the co-stimulatory/-inhibitory B7-family, exerts immunosuppressive and pro-tumorigenic functions in various cancer types and is under evaluation in ongoing clinical trials. Unfortunately, interaction partner(s) remain unknown which restricts the druggability. Methods: Aiming to identify potential binding partner(s) of B7-H3, a yeast two-hybrid and a mass spectrometry screen were performed. Potential candidates were evaluated by bimolecular fluorescence complementation (BiFC) assay, co-immunoprecipitation (co-IP), and functionally in a 3H-thymidine proliferation assay of Jurkat cells, a T-cell lineage cell line. Prognostic value of angio-associated migratory cell protein (AAMP) and B7-H3 expression was evaluated in isocitrate dehydrogenase 1 wildtype (IDH1wt) glioblastoma (GBM) patients from The Cancer Genome Atlas (TCGA)-GBM cohort. Results: Of the screening candidates, CD164, AAMP, PTPRA, and SLAMF7 could be substantiated via BiFC. AAMP binding could be further confirmed via co-IP and on a functional level. AAMP was ubiquitously expressed in glioma cells, immune cells, and glioma tissue, but did not correlate with glioma grade. Finally, an interaction between AAMP and B7-H3 could be observed on expression level, hinting toward a combined synergistic effect. Conclusions: AAMP was identified as a novel interaction partner of B7-H3, opening new possibilities to create a targeted therapy against the pro-tumorigenic costimulatory protein B7-H3.