Identification of PD-L2, B7-H3 and CTLA-4 immune checkpoint proteins in genetic subtypes of meningioma

Abstract

Meningioma is the most common brain tumor in adults. Surgical resection remains the primary treatment. No chemotherapy exists. However, gene mutations now could explain ~ 80% of meningioma and targeted therapies based on these are being investigated. Furthermore, with the recent discovery of PD-L1 in malignant meningioma, clinical trials using immunotherapy have commenced. Here, we report for the first time the expression profiles of immune checkpoint proteins PD-L2, B7-H3 and CTLA-4 in meningioma and their association to common gene mutations. PD-L2 and B7-H3 expression was significantly greater than all immune checkpoint proteins studied, and particularly elevated in patients with gene mutations affecting the PI3K/AKT/mTOR pathway. CTLA-4 expressing CD3+ lymphocytes were observed in atypical and malignant meningioma and tumors harboring a PIK3CA or SMO mutation. These results identify novel targets for immunotherapy irrespective of grade and distinguish potential patient populations based on genetic classification for stratification into checkpoint inhibitor clinical trials.

Keywords: B7-H3; CTLA-4; Meningioma; PD-L1; PD-L2; PI3K/AKT/mTOR; brain tumor; immune checkpoint protein; immunotherapy.

Figure 1.

Immune checkpoint protein expression in meningioma tissues according to WHO grade and genetic subtype. (A) Quantities of 10 common immune checkpoint proteins in meningioma tissues according to WHO grade (n = 22). Amounts of B7-H3 and PD-L2 proteins are significantly greater than all other proteins analyzed. (B) Expression of PD-L2, B7-H3 and CTLA-4 for each case (n = 20) is quantified according to WHO grade and gene mutation subtype. (C) Representative scans of a multiplex ELISA array for a single protein standard used in the generation of a protein quantification standard curve and also a meningioma tissue protein sample. *** P-value < 0.001. Bars represent mean protein quantity in pg/mg of meningioma tissue. Error bars represent SEM.

Figure 2.

Fluorescence immunohistochemistry of B7-H3, PD-L2, and CTLA-4 in meningioma and lateral temporal cortex tissues. (A) Representative images of B7-H3, PD-L2, and CTLA-4 expression (red) in meningioma specimens and lateral temporal cortex control. Staining severity was determined semi-quantitatively as outlined in methods. Blue represents nuclear DAPI stain. All images were taken with Olympus BX51 fluorescence microscope at 40x magnification with scale bar measuring 50 µm. (B) Summary of CD3, B7-H3, PD-L2, and CTLA-4 expression in 21 meningioma patients (M#) and 2 lateral temporal cortex controls (C#). Staining severity (low, moderate and high expression) follows semi-quantitative characterization as outlined in methods.

Figure 3.

Fluorescence immunohistochemistry co-staining of CD3 together with B7-H3, PD-L2, and CTLA-4 in meningioma tissues. Representative images of DAPI (blue), CD3 (green) and B7-H3, PD-L2 and CTLA-4 (red) stains at 40x magnification (Olympus BX51). Bottom panel shows overlay image of DAPI, CD3 and checkpoint protein. Scale bar measures 50 µm. Mild expression is not shown in the figure as B7-H3 and CTLA-4 showed no mild staining.

Figure 4.

Effects of edema on immune checkpoint expression and correlation with tumor size, grade and genetic subtype. Correlation analysis of edema severity with tumor size (A) and tumor WHO grade (B). (C) Expression of PD-L2 and B7-H3 is quantified according to severity of edema. (D) Edema severity of each patient according to genetic mutation subtype. (Edema severity in A and B; 0 = nil, 1 = mild, 2 = moderate, 3 = severe). (E) Representative axial T₁ post – Gd MR images acquired at 1.5T and associated levels of immune checkpoint proteins from 5 patients.

Figure 5.

Oncogenic pathways and drug targets important to meningioma and immune checkpoint protein regulation. Key pathways involved in regulating checkpoint proteins in meningioma are highlighted in yellow. Drug therapies in clinical trial or FDA approved towards targets are in red. * Represents mutations in encoding gene is common in meningioma.