No abstract

The use of gene expression profiling (GEP) in cancer management is rising, as GEP can be used for disease classification and diagnosis, tailoring treatment to underlying genetic determinants of pharmacological response, monitoring of therapy response, and prognosis. However, the reliability of GEP heavily depends on the input of RNA in sufficient quantity and quality. This highlights the need for standard procedures to ensure best practices for RNA extraction from often small tumor biopsies with variable tissue handling. We optimized an RNA extraction protocol from fresh-frozen (FF) core needle biopsies (CNB) from breast cancer patients and from formalin-fixed paraffin-embedded (FFPE) tissue when FF CNB did not yield sufficient RNA. Methods to avoid ribonucleases andto homogenize or to deparaffinize tissues and the impact of tissue composition on RNA extraction were studied. Additionally, RNA’s compatibility with the nanoString nCounter® technology was studied. This technology platform enables GEP using small RNA fragments. After optimization of the protocol, RNA of high quality and sufficient quantity was obtained from FF CNB in 92% of samples. For the remaining 8% of cases, FFPE material prepared by the pathology department was used for RNA extraction. Both resulting RNA end products are compatible with the nanoString nCounter® technology.

Dendritic cell (DC)-maturation stimuli determine the potency of these antigen-presenting cells and, therefore, the quality of the T-cell response. Here we describe that the maturation of DCs via TriMix mRNA, encoding CD40 ligand, a constitutively active variant of toll-like receptor 4 and the co-stimulatory molecule CD70, enables an antibacterial transcriptional program. Besides, we further show that the DCs are redirected into an antiviral transcriptional program when CD70 mRNA in TriMix is replaced with mRNA encoding interferon-gamma and a decoy interleukin-10 receptor alpha, forming a four-component mixture referred to as TetraMix mRNA. The resulting TetraMixDCs show a high potential to induce tumor antigen-specific T cells within bulk CD8+ T cells. Tumor-specific antigens (TSAs) are emerging and attractive targets for cancer immunotherapy. As T-cell receptors recognizing TSAs are predominantly present on naive CD8+ T cells (TN), we further addressed the activation of tumor antigen-specific T cells when CD8+ TN cells are stimulated by TriMixDCs or TetraMixDCs. In both conditions, the stimulation resulted in a shift from CD8+ TN cells into tumor antigen-specific stem cell-like memory, effector memory and central memory T cells with cytotoxic capacity. These findings suggest that TetraMix mRNA, and the antiviral maturation program it induces in DCs, triggers an antitumor immune reaction in cancer patients.

The purpose of this study was to test a newly developed decontamination and fluidization kit for processing respiratory specimens for the detection of mycobacteria: the Myco-TB procedure (developed by Copan (Brescia, Italy)). This technique was compared with the Zephiran decontamination method in use in our hospital. Respiratory specimens (n = 387: 130 endotracheal/bronchial aspirates, 172 bronchoalveolar lavages and 55 sputa) submitted to the University Hospital of Brussels between January 2016 and March 2017 were included. All samples were divided into two aliquots: one was subjected to the Myco-TB method and one to the Zephiran technique prior to culture. The sensitivities for culture for the Zephiran technique on solid media, the Myco-TB method on solid media and Myco-TB combined with the MGIT™ system were respectively 67%, 87% and 89%. The contamination rates were 22% with both the Zephiran and Myco-TB method on solid media and only 4% with the Myco-TB kit combined with the MGIT™ system. For direct microscopy, the sensitivities of the Zephiran method and the Myco-TB method were equal (40%) when the centrifugation time was 20 min. The Myco-TB decontamination method is easy and rapid to perform. It is more sensitive for culture as compared to the Zephiran method and gives lower contamination levels when combined with the MGIT™ technique. When increasing the centrifugation step to 20 min, the sensitivity of direct microscopy is equal to the Zephiran method.