Alonso Barrantes-Freer

Colony‐stimulating factor 1 receptor (CSF1R)‐associated leukoencephalopathy (CSF1R‐LE) is a fatal genetic white matter disease, whose onset is most commonly in mid‐age adulthood, and is characterized by rapid neurological decline and premature death within a few years after disease onset. CSF1R is exclusively expressed by microglia in the parenchyma of the adult central nervous system, thus defining CSF1R‐LE as a paradigmatic primary microgliopathy. However, the cellular alterations that drive the sudden onset and rapid progression of the disease remain largely unknown. Therefore, the investigation of microglia phenotypes in human histopathological tissue, particularly considering the spatial evolution of the pathology, can provide crucial insights into the disease‐causing mechanisms. In this study, we (immuno‐) histochemically characterized myeloid cell pathology in relation to the extent of damage to the axon‐myelin unit in five autopsy cases of CSF1R‐LE. A particular focus was on the relationship between microglia phenotypes and the pronounced iron accumulation observed in this disease. Our findings provide evidence that the early loss of homeostatic microglia and inflammatory activation of remaining myeloid cells are key components of areas with mild tissue pathology. Furthermore, we demonstrate a marked and progressive accumulation of iron within myeloid cells paralleling increasing white matter damage. This process is associated with ongoing myelin degeneration and occurs in the apparent absence of efficient remyelination. In summary, our study reveals stage‐specific myeloid cell pathology in CSF1R‐LE and highlights a specific spatial heterogeneity that should be considered when studying this disease. Moreover, our findings suggest that cellular iron accumulation may not only contribute to disease progression but may also represent a promising and clinically accessible biomarker for CSF1R‐LE.

BeadChip array‐based DNA methylation profiling has been recognized by the World Health Organization (WHO) as a key diagnostic tool for brain tumor classification. While its diagnostic utility has been well established, data on technical reproducibility, interlaboratory comparability, and data interpretation under real‐world diagnostic conditions remain limited. Bridging this gap, we here report the results of an international proficiency test using the Infinium MethylationEPIC v2.0 platform and the corresponding Brain Tumor Classifier version 12.8. Tissue slides of eight FFPE brain tumor samples, covering a representative range of CNS tumor entities, were distributed among 24 laboratories in 10 different countries. Participants were asked to report methylation classes and copy number variation (CNV) profiles. Pre‐array workflows were left to local procedures and results had to be submitted within 15 working days. Technical data reproducibility was high with a median pairwise beta‐value correlation of 0.99 (range 0.93–1.0). In general, participating centers generated high‐quality data, reflected by consistently low detection p ‐values (<0.01). Eighteen of the 24 participating centers (75%) successfully passed the test. Of the six centers that failed the test, two laboratories experienced technical issues that led to misclassification of individual cases and contributed to incorrect CNV reporting. Four additional centers showed substantial discrepancies in the interpretation of diagnostically highly relevant CNVs, whereas methylation classification was not impaired. While accurate DNA quantification proved to be an important pre‐array step, the use of the DNA restoration kit had only minor influence on overall results. Taken together, our interlaboratory performance testing on EPIC v2.0 CNS tumor profiling confirms high reproducibility of tumor classification but reveals the need for harmonized CNV reporting.

PolySia is not associated with CD3- or CD34-positive cells

Antibody array detection of cytokines and chemokines

PolySia is associated with GFAP-positive cells

PolySia on CD11c-positive and on CD45-positive, IBA1-negative cells

Supplementary Table S1.Demographic and clinical parameters of GB patient cohorts.Supplementary Table S2.Characteristics of the two cohorts of n = 70 (MHH) and n = 100 (UMG) GB patients stratified for the presence of a functional SIGLEC16 allele (A) or for the occurrence of polySia on tumor cells (B).Supplementary Table S3.Cox proportional hazards regression analysis of covariates for SIGLEC16 and polySia status in relation to OS (MHH and UMG cohorts combined).Supplementary Table S4.Median age of GB cases stratified for SIGLEC16 genotype and polySia on tumor cells.Supplementary Table S5.OS analysis of GB cases receiving (A) or not receiving gross total resection (B) stratified for SIGLEC16 genotype and polySia on tumor cells (MHH and UMG cohorts combined).Supplementary Table S6.OS analysis of GB cases receiving (A) or not receiving chemotherapy (B) stratified for SIGLEC16 genotype and polySia on tumor cells (MHH cohort only).Supplementary Table S7.OS analysis of GB cases receiving gross total resection and chemotherapy stratified for SIGLEC16 genotype and polySia on tumor cells (MHH and UMG cohorts combined, n=101).Supplementary Table S8.OS analysis of IDH-wildtype GB cases (UMG cohort) stratified for SIGLEC16 genotype and polySia on tumor cells.Supplementary Table S9.OS analysis of UMG cohort stratified for MGMT gene promoter methylation and SIGLEC16 genotype, polySia on tumor cells, CD163 and CD74 status.Supplementary Table S10.Characteristics of the two cohorts of n = 70 (MHH) and n = 100 (UMG) GB patients stratified for CD74low and CD74high.Supplementary Table S11. Median age at resection of GB cases stratified for CD163 to CD68 (A) or CD74 to IBA1 ratios (B) alone or in combination with the SIGLEC16 genotype.Supplementary Table S12. OS analysis of GB cases receiving (A) or not receiving gross total resection (B) stratified for CD163 to CD68 or CD74 to IBA1 ratios alone or in combination with the SIGLEC16 genotype (MHH and UMG cohorts combined).Supplementary Table S13. OS analysis of GB cases receiving (A) or not receiving chemotherapy (B) stratified for CD163 to CD68 or CD74 to IBA1 ratios alone or in combination with the SIGLEC16 genotype (MHH cohort only).Supplementary Table S14.OS analysis of GB cases receiving both gross total resection and chemotherapy stratified for CD163 to CD68 or CD74 to IBA1 ratios alone or in combination with the SIGLEC16 genotype (MHH and UMG cohorts combined, n=101).

OS analysis of the two GB cohorts individually stratified for CD163 to CD68 or CD74 to IBA1 ratios

PolySia on GB tumor cells is associated with NCAM