The histological analysis of adrenal tumors is difficult and requires great expertise. Tissue microRNA (miRNA) expression is distinct between benign and malignant tumors of several organs and can be useful for diagnostic purposes. MiRNAs are stable and their expression can be reliably reproduced from archived formalin-fixed, paraffin-embedded (FFPE) tissue blocks. Our purpose was to assess the potential applicability of combinations of literature-based miRNAs as markers of adrenocortical malignancy. Archived FFPE tissue samples from 10 adrenocortical carcinoma (ACC), 10 adrenocortical adenoma (ACA) and 10 normal adrenal cortex samples were analyzed in a discovery cohort, while 21 ACC and 22 ACA patients were studied in a blind manner in the validation cohort. The expression of miRNA was determined by RT-qPCR. Machine learning and neural network-based methods were used to find the best performing miRNA combination models. To evaluate diagnostic applicability, ROC-analysis was performed. We have identified three miRNA combinations (hsa-miR-195 + hsa-miR-210 + hsa-miR-503; hsa-miR-210 + hsa-miR-375 + hsa-miR-503 and hsa-miR-210 + hsa-miR-483-5p + hsa-miR-503) as unexpectedly good predictors to determine adrenocortical malignancy with sensitivity and specificity both of over 90%. These miRNA panels can supplement the histological examination of removed tumors and could even be performed from small volume adrenal biopsy samples preoperatively.

Cells of the monocyte macrophage lineage form multinucleated giant cells (GCs) by fusion, which may express some cell cycle markers. By using a comprehensive marker set, here we looked for potential replication activities in GCs, and investigated whether these have diagnostic or clinical relevance in giant cell tumor of bone (GCTB). GC rich regions of 10 primary and 10 first recurrence GCTB cases were tested using immunohistochemistry in tissue microarrays. The nuclear positivity rate of the general proliferation marker, replication licensing, G1/S-phase, S/G2/M-phase, mitosis promoter, and cyclin dependent kinase (CDK) inhibitor reactions was analyzed in GCs. Concerning Ki67, moderate SP6 reaction was seen in many GC nuclei, while B56 and Mib1 positivity was rare, but the latter could be linked to more aggressive (p = 0.012) phenotype. Regular MCM6 reaction, as opposed to uncommon MCM2, suggested an initial DNA unwinding. Early replication course in GCs was also supported by widely detecting CDK4 and cyclin E, for the first time, and confirming cyclin D1 upregulation. However, post-G1-phase markers CDK2, cyclin A, geminin, topoisomerase-2a, aurora kinase A, and phospho-histone H3 were rare or missing. These were likely silenced by upregulated CDK inhibitors p15^INK4b, p16^INK4a, p27^KIP1, p53 through its effector p21^WAF1 and possibly cyclin G1, consistent with the prevention of DNA replication. In conclusion, the upregulation of known and several novel cell cycle progression markers detected here clearly verify early replication activities in GCs, which are controlled by cell cycle arresting CDK inhibitors at G1 phase, and support the functional maturation of GCs in GCTB.

Differentiation of adrenocortical adenoma (ACA) and carcinoma (ACC) is often challenging even in the histological analysis. Circular RNAs (circRNAs) belonging to the group of non-coding RNAs have been implicated as relevant factors in tumorigenesis. Our aim was to explore circRNA expression profiles in adrenocortical tumors by next-generation sequencing followed by RT-qPCR validation. Archived FFPE (formalin-fixed, paraffin embedded) including 8 ACC, 8 ACA and 8 normal adrenal cortices (NAC) were used in the discovery cohort. For de novo and known circRNA expression profiling, a next-generation sequencing platform was used. CIRI2, CircExplorer2, AutoCirc bioinformatics tools were used for the discovery of circRNAs. The top five most differentially circRNAs were measured by RT-qPCR in an independent validation cohort (10 ACC, 8 ACA, 8 NAC). In silico predicted, interacting microRNAs potentially sponged by differentially expressed circRNAs were studied by individual RT-qPCR assays. We focused on overexpressed circRNAs here. Significantly differentially expressed circRNAs have been revealed between the cohorts by NGS. Only circPHC3 could be confirmed to be significantly overexpressed in ACC, ACA vs. NAC samples by RT-qPCR. We could not observe microRNA expression changes fully corresponding to our sponging hypothesis. To the best of our knowledge, our study is the first to investigate circRNAs in adrenocortical tumors. Further studies are warranted to explore their biological and diagnostic relevance.