Differentially Tolerized Mouse Antigen Presenting Cells Share a Common miRNA Signature Including Enhanced mmu-miR-223-3p Expression Which Is Sufficient to Imprint a Protolerogenic State.

Dendritic cells (DCs) are pivotal for the induction and maintenance of antigen-specific tolerance and immunity. miRNAs mediate post-transcriptional gene regulation and control in part the differentiation and stimulation-induced immunogenic function of DCs. However, the relevance of miRNAs for the induction and maintenance of a tolerogenic state of DCs has scarcely been highlighted yet. We differentiated mouse bone marrow cells to conventional/myeloid DCs or to tolerogenic antigen presenting cells (APCs) by using a glucocorticoid (dexamethasone) or interleukin-10, and assessed the miRNA expression patterns of unstimulated and LPS-stimulated cell populations by array analysis and QPCR. Differentially tolerized mouse APCs convergingly down-regulated a set of miRNA species at either state of activation as compared with the corresponding control DC population (mmu-miR-9-5p, mmu-miR-9-3p, mmu-miR-155-5p). These miRNAs were also upregulated in control DCs in response to stimulation. In contrast, miRNAs that were convergingly upregulated in both tolerized APC groups at stimulated state (mmu-miR-223-3p, mmu-miR-1224-5p) were downregulated in control DCs in response to stimulation. Overexpression of mmu-miR-223-3p in DCs was sufficient to prevent stimulation-associated acquisition of potent T cell stimulatory capacity. Overexpression of mmu-miR-223-3p in a DC line resulted in attenuated expression of known (Cflar, Rasa1, Ras) mRNA targets of this miRNA species shown to affect pathways that control DC activation. Taken together, we identified sets of miRNAs convergingly regulated in differentially tolerized APCs, which may contribute to imprint stimulation-resistant tolerogenic function as demonstrated for mmu-miR-223-3p. Knowledge of miRNAs with protolerogenic function enables immunotherapeutic approaches aimed to modulate immune responses by regulating miRNA expression.

Multi-center evaluation of the novel fully-automated PCR-based Idylla™ BRAF Mutation Test on formalin-fixed paraffin-embedded tissue of malignant melanoma.

The advent of BRAF-targeted therapies led to increased survival in patients with metastatic melanomas harboring a BRAF V600 mutation (implicated in 46-48% of malignant melanomas). The Idylla(™) System (Idylla(™)), i.e., the real-time-PCR-based Idylla(™) BRAF Mutation Test performed on the fully-automated Idylla(™) platform, enables detection of the most frequent BRAF V600 mutations (V600E/E2/D, V600K/R/M) in tumor material within approximately 90 min and with 1% detection limit. Idylla(™) performance was determined in a multi-center study by analyzing BRAF mutational status of 148 archival formalin-fixed paraffin-embedded (FFPE) tumor samples from malignant melanoma patients, and comparing Idylla(™) results with assessments made by commercial or in-house routine diagnostic methods. Of the 148 samples analyzed, Idylla(™) initially recorded 7 insufficient DNA input calls and 15 results discordant with routine method results. Further analysis learned that the quality of 8 samples was insufficient for Idylla(™) testing, 1 sample had an invalid routine test result, and Idylla(™) results were confirmed in 10 samples. Hence, Idylla(™) identified all mutations present, including 7 not identified by routine methods. Idylla(™) enables fully automated BRAF V600 testing directly on FFPE tumor tissue with increased sensitivity, ease-of-use, and much shorter turnaround time compared to existing diagnostic tests, making it a tool for rapid, simple and highly reliable analysis of therapeutically relevant BRAF mutations, in particular for diagnostic units without molecular expertise and infrastructure.