PhD Scientific Days 2025

Budapest, 7-9 July 2025

Poster Session II. - W: Conservative Medicine

Pitfalls of circulating microRNA-based research in adrenal tumors

Name of the presenter

Vékony Bálint

Institute/workplace of the presenter

Semmelweis University, Department of Internal Medicine and Oncology, Department of Endocrinology

Authors

Bálint Vékony1,2, Gábor Nyirő1,2,3, Henriett Butz3,4,5, Bálint Kende Szeredás1,2, Viktória Tóth6,7, Péter Ferdinándy6,7, Attila Patócs3,4,5, Péter Igaz1,2

1: Department of Internal Medicine and Oncology, Semmelweis University, Budapest, Hungary
2: Department of Endocrinology, Semmelweis University, Budapest, Hungary
3: Department of Laboratory Medicine, Semmelweis University, Budapest, Hungary
4: HUN-REN-SU, Hereditary Tumours Research Group, Budapest, Hungary
5: National Institute of Oncology, Department of Molecular Genetics and the National Tumour Biology Laboratory, Budapest, Hungary
6: Department of Pharmacology and Pharmacotherapy, Semmelweis University, Budapest, Hungary
7: Center for Pharmacology, Drug Research and Development, Semmelweis University, Budapest, Hungary

Text of the abstract

Introduction: Differentiating between benign and malignant adrenocortical tumors has major clinical relevance. Recent studies have highlighted the potential of circulating microRNAs (miRNAs) as biomarkers for malignant adrenocortical carcinoma (ACC). However, there are many difficulties with their use, mainly standardization.
Aims: Our aim was to compare the interchangeability of real-time quantitative polymerase chain reaction (qPCR) and digital PCR (dPCR) in measuring circulating miRNAs, and to investigate whether the use of K2 or K3 EDTA anticoagulants may influence the results.
Methods: Peripheral blood samples were taken simultaneously into K2 and K3 EDTA collection tubes, from 20 individuals. After sample procession, three miRNAs associated with ACC (hsa-miR-483-5p, -210-3p, -21-5p) as well as two controls (-miR-16-5p, cel-miR-39-3p) were analyzed utilizing RT-qPCR and dPCR. Data obtained were compared by the following statistical methods: Spearman’s rank correlation, paired t tests and BlandAltman analysis.
Results: qPCR and dPCR results show a significant correlation (p values between 0.0072 and 0.049) in K2-EDTA samples when comparing DCt values and copy numbers. However, proportional biases relating to low and high miRNA expression were observed between the two methods. In qPCR measurements, K3-EDTA results showed higher standard deviations (average SD for K2 samples was 0.91 while for K3 samples 1.1). When comparing raw Ct values, only miR-483-5p was found to be significantly different, while in case of DCt values, every miRNA except miR-483-5p was significantly different. dPCR results were not affected by the different anticoagulants.
Conclusions: dPCR and qPCR are not easily interchangeable, especially for rare or abundant miRNAs, making crossvalidation difficult. The choice of EDTA could potentially influence qPCR results, highlighting the need for standardized protocols.
Funding: The authors acknowledge support from the Hungarian National Research, Development and Innovation Office (NKFIH) (grants K146906 to PI) and TKP2021-EGA-24 from the National Research, Development and Innovation Fund by the Ministry of Innovation and Technology of Hungary financed under the [TKP2021-EGA] funding scheme and also supported by the 2024-2.1.1.-EKÖP-2020-00004 University research scholarship programme of the Ministry for Culture and Innovation.