Monocytes carrying GFAP detect glioma, brain metastasis and ischaemic stroke, and predict glioblastoma survival

Abstract

Diagnosis and monitoring of primary brain tumours, brain metastasis and acute ischaemic stroke all require invasive, burdensome and costly diagnostics, frequently lacking adequate sensitivity, particularly during disease monitoring. Monocytes are known to migrate to damaged tissues, where they act as tissue macrophages, continuously scavenging, phagocytizing and digesting apoptotic cells and other tissue debris. We hypothesize that upon completion of their tissue-cleaning task, these tissue macrophages might migrate via the lymph system to the bloodstream, where they can be detected and evaluated for their phagolysosomal contents. We discovered a blood monocyte subpopulation carrying the brain-specific glial fibrillary acidic protein in glioma patients and in patients with brain metastasis and evaluated the diagnostic potential of this finding. Blood samples were collected in a cross-sectional study before or during surgery from adult patients with brain lesions suspected of glioma. Together with blood samples from healthy controls, these samples were flowing cytometrically evaluated for intracellular glial fibrillary acidic protein in monocyte subsets. Acute ischaemic stroke patients were tested at multiple time points after onset to evaluate the presence of glial fibrillary acidic protein-carrying monocytes in other forms of brain tissue damage. Clinical data were collected retrospectively. High-grade gliomas (N = 145), brain metastasis (N = 21) and large stroke patients (>100 cm³) (N = 3 versus 6; multiple time points) had significantly increased frequencies of glial fibrillary acidic protein+CD16+ monocytes compared to healthy controls. Based on both a training and validation set, a cut-off value of 0.6% glial fibrillary acidic protein+CD16+ monocytes was established, with 81% sensitivity (95% CI 75-87%) and 85% specificity (95% CI 80-90%) for brain lesion detection. Acute ischaemic strokes of >100 cm³ reached >0.6% of glial fibrillary acidic protein+CD16+ monocytes within the first 2-8 h after hospitalization and subsided within 48 h. Glioblastoma patients with >20% glial fibrillary acidic protein+CD16+ non-classical monocytes had a significantly shorter median overall survival (8.1 versus 12.1 months). Our results and the available literature, support the hypothesis of a tissue-origin of these glial fibrillary acidic protein-carrying monocytes. Blood monocytes carrying glial fibrillary acidic protein have a high sensitivity and specificity for the detection of brain lesions and for glioblastoma patients with a decreased overall survival. Furthermore, their very rapid response to acute tissue damage identifies large areas of ischaemic tissue damage within 8 h after an ischaemic event. These studies are the first to report the clinical applicability for brain tissue damage detection through a minimally invasive diagnostic method, based on blood monocytes and not serum markers, with direct consequences for disease monitoring in future (therapeutic) studies and clinical decision making in glioma and acute ischaemic stroke patients.

Keywords: acute ischemic stroke; glioma; metastatic CNS tumour; minimal invasive diagnostics; neuroimmunology.

Graphical Abstract

Figure 1

Detection of GFAP+ macrophages in brain tissue and in monocytes from patients suspected of glioma. (A) Dissociated glioma tissue contains CD68+ (green) and GFAP+ (magenta) cells. (B) Flow cytometric dot plots of blood monocytes from two patients suspected of glioma lesions and their GFAP expression levels. (C) Flow cytometric dot plot of GFAP expression in monocyte subsets in a representative example of a healthy control. White bar represents scale of the microscopy images. Percentage of GFAP+ cells (B, C). Dot plots show classical (red), intermediate (light blue) and non-classical (dark blue) monocytes.

Figure 2

Distribution of GFAP+CD16+ monocytes in blood in brain lesions versus healthy controls and relation to OS in GBM patients. (A) Violin plot with median, quartiles and individual percentages of GFAP+CD16+ monocytes in blood of patients with different types of glioma, brain metastasis, other brain tumours and healthy controls (Kruskal–Wallis). (B) ROC curve with area under the curve for presence of a brain lesion, with GFAP+CD16+ monocytes as predictor in the training set (green dotted line (N = 82) and validation set (dark green line, N = 157). (C) Survival plot of GBM patients with GFAP+CD14−CD16+ monocytes below (light blue) or over 20% (dark blue) of GFAP-positivity. Ticks mark censored cases, Log-rank test. AUC = area under the curve; Oligo = oligodendroglioma; ROC = receiver operating characteristic; ** P < 0.01, *** P < 0.005, **** P < 0.001.

Figure 3

Monitoring of percentage of GFAP+CD16+ monocytes in blood during the first 96 h following onset of stroke symptoms. Relative numbers of GFAP+CD16+ monocytes in blood are shown for 9 AIS patients after stroke onset, coloured differently according to the size of their corresponding ischaemic lesion (in cm³): red ≥100 cm³; blue and grey: <100 cm³. The dotted line represents the 0.6% GFAP+CD16+ monocyte CO.