Optimized tumour infiltrating lymphocyte assessment for triple negative breast cancer prognostics.

The tumour microenvironment has been shown to be a valuable source of prognostic information for different cancer types. This holds in particular for triple negative breast cancer (TNBC), a breast cancer subtype for which currently no prognostic biomarkers are established. Although different methods to assess tumour infiltrating lymphocytes (TILs) have been published, it remains unclear which method (marker, region) yields the most optimal prognostic information. In addition, to date, no objective TILs assessment methods are available. For this proof of concept study, a subset of our previously described TNBC cohort (n = 94) was stained for CD3, CD8 and FOXP3 using multiplex immunohistochemistry and subsequently imaged by a multispectral imaging system. Advanced whole-slide image analysis algorithms, including convolutional neural networks (CNN) were used to register unmixed multispectral images and corresponding H&E sections, to segment the different tissue compartments (tumour, stroma) and to detect all individual positive lymphocytes. Densities of positive lymphocytes were analysed in different regions within the tumour and its neighbouring environment and correlated to relapse free survival (RFS) and overall survival (OS). We found that for all TILs markers the presence of a high density of positive cells correlated with an improved survival. None of the TILs markers was superior to the others. The results of TILs assessment in the various regions did not show marked differences between each other. The negative correlation between TILs and survival in our cohort are in line with previous studies. Our results provide directions for optimizing TILs assessment methodology.

IL-1ß processing in mechanical ventilation-induced inflammation is dependent on neutrophil factors rather than caspase-1.

PURPOSE: Mechanical ventilation can cause ventilator-induced lung injury, characterized by a sterile inflammatory response in the lungs resulting in tissue damage and respiratory failure. The cytokine interleukin-1ß (IL-1ß) is thought to play an important role in the pathogenesis of ventilator-induced lung injury. Cleavage of the inactive precursor pro-IL-1ß to form bioactive IL-1ß is mediated by several types of proteases, of which caspase-1, activated within the inflammasome, is the most important. Herein, we studied the roles of IL-1ß, caspase-1 and neutrophil factors in the mechanical ventilation-induced inflammatory response in mice. METHODS: Untreated wild-type mice, IL-1αß knockout and caspase-1 knockout mice, pralnacasan (a selective caspase-1 inhibitor)-treated mice, anti-keratinocyte-derived chemokine (KC)-treated mice and cyclophosphamide-treated neutrophil-depleted wild-type mice were ventilated using clinically relevant ventilator settings (tidal volume 8 ml/kg). The lungs and plasma were collected to determine blood gas values, cytokine profiles and neutrophil influx. RESULTS: Mechanical ventilation resulted in increased pulmonary concentrations of IL-1ß and KC and increased pulmonary neutrophil influx compared with non-ventilated mice. Ventilated IL-1αß knockout mice did not demonstrate this increase in cytokines. No significant differences were observed between wild-type and caspase-1-deficient or pralnacasan-treated mice. In contrast, in anti-KC antibody-treated mice and neutropenic mice, inflammatory parameters decreased in comparison with ventilated non-treated mice. CONCLUSIONS: Our results illustrate that IL-1 is indeed an important cytokine in the inflammatory cascade induced by mechanical ventilation. However, the inflammasome/caspase-1 appears not to be involved in IL-1ß processing in this type of inflammatory response. The attenuated inflammatory response observed in ventilated anti-KC-treated and neutropenic mice suggests that IL-1ß processing in mechanical ventilation-induced inflammation is mainly mediated by neutrophil factors.