Purpose Resection of high-grade gliomas has been considerably improved by 5-aminolevulinic acid (5-ALA). However, not all neurobiological properties of 5-ALA are fully understood. Specifically, potential differences in immune infiltration have not been conclusively examined, despite recent reports that immune cells might play a role. Thus, we here provide a systematic mapping of immune infiltration of different 5-ALA fluorescence levels. Methods Tumor-associated macrophages (CD68, CD163), cytotoxic T cells (CD8), and regulatory T cells (FoxP3) were quantified via three methods. First, data from The Cancer Genome Atlas (TCGA) of 172 patients was examined for correlations between 5-ALA fluorescence-related mRNA expression signatures and immune markers. Second, as classical histology, 508 stained slides from 39 high-grade glioma patients were analysed semi-quantitatively by two independent reviewers, generating 1016 data points. Third, digital image analysis was performed with automated scanning and algorithm-based cell quantification. Results TCGA mRNA data from 172 patients showed a direct, significant correlation between 5-ALA signatures and immune markers (p < 0.001). However, we were not able to confirm this finding in the here studied initial set of 39 patient histologies where we found a comparable immune infiltration in different fluorescence levels. Digital image analysis correlated excellently with standard histology. Conclusion With mapping the immune infiltration pattern of different 5-ALA categories, we are adding fundamental basic insights to the field of 5-ALA and glioma biology. The observation that a significant correlation in TCGA data did not fully translate to detectable differences in immune infiltration in first histology data warrants further investigation in larger cohorts.

Background Fluorescence-guided surgery with 5-aminolevulinic acid (5-ALA) is a widely used technique to conduct maximum safe resection of high-grade gliomas (HGG). 5-ALA accumulates in malignant tumor tissue where it is metabolized to Protoporphyrin IX (PpIX), an agent with fluorescence properties. It helps neurosurgeons to distinguish between tumor-infiltrated tissue and healthy brain parenchyma. Even though fluorescence-guided surgery is clinically well established, the biological properties of different fluorescence levels are not comprehensively studied yet. A relevant current gap in that respect is the pattern of immune cell infiltration in fluorescent versus non-fluorescent tissue samples. In light of future research, reliable, standardized histopathology methods that allow high-throughput analysis are desirable and digital pathology with automated image analysis is an attractive option to explore. Material and Methods 128 formalin-fixed paraffin-embedded (FFPE) tissue blocks of 39 patients who underwent fluorescence-guided surgery of a HGG were included. Samples were selected based on their documented 5-ALA fluorescence intensity status (strong, vague, negative). Microtome-cut sections of the tissue were stained with antibodies against CD8, CD68, CD163 and FOX P3, representing immune cell populations of specific interest (cytotoxic T cells, glioma-associated macrophages, regulatory T cells). A total of 512 stained slides were then available for assessment. In addition to a classical, semi-quantitative analysis by two independent human reviewers, the immune infiltration intensity was measured via automated image analysis with the digital pathology software QuPath Version 0.3.2. Results Across all stained FFPE samples CD68 showed the overall highest intensity, followed by CD163. CD8 and FoxP3 showed generally lower average intensities. In 5-ALA negative and positive high-grade glioma samples the immune cell infiltration pattern was the same. Quantitative automatic digital pathology correlated well with the classical human histopathological analysis for the majority of markers evaluated. Conclusion We successfully explored and established novel digital pathology technologies for the study of immune cell infiltration patterns in neurooncology, specifically in the context of fluorescence-guided resection. Leveraging this platform could allow parallelized and high-throughput analysis of immune cell infiltration also in other contexts.