A Rapid Robust Method for Subgrouping Non-NF2 Meningiomas According to Genotype and Detection of Lower Levels of M2 Macrophages in AKT1 E17K Mutated Tumours

Abstract

The majority of meningiomas are grade I, but some grade I tumours are clinically more aggressive. Recent advances in the genetic study of meningiomas has allowed investigation into the influence of genetics on the tumour microenvironment, which is important for tumorigenesis. We have established that the endpoint genotyping method Kompetitive Allele Specific PCR (KASP™) is a fast, reliable method for the screening of meningioma samples into different non-NF2 mutational groups using a standard real-time PCR instrument. This genotyping method and four-colour flow cytometry has enabled us to assess the variability in the largest immune cell infiltrate population, M2 macrophages (CD45+HLA-DR+CD14+CD163+) in 42 meningioma samples, and to suggest that underlying genetics is relevant. Further immunohistochemistry analysis comparing AKT1 E17K mutants to WHO grade I NF2-negative samples showed significantly lower levels of CD163-positive activated M2 macrophages in meningiomas with mutated AKT1 E17K, signifying a more immunosuppressive tumour microenvironment in NF2 meningiomas. Our data suggested that underlying tumour genetics play a part in the development of the immune composition of the tumour microenvironment. Stratifying meningiomas by mutational status and correlating this with their cellular composition will aid in the development of new immunotherapies for patients.

Basis for Immunotherapy for Treatment of Meningiomas

“…Other mutations like TRAF7, AKT1, KLF4, SMO, and PIK3CA were more recently described ( 27 , 28 ) and may represent therapeutic targets ( 29 – 31 ). In one particular study, grade I meningiomas harboring AKT1 mutations had predominantly M2-subtype infiltrating macrophages, indicating a locally immunosuppressed tumor microenvironment ( 32 ). Additionally, it has been found that tumors initially diagnosed as WHO grade II meningiomas commonly have loss of NF2 with alterations in SMARCB1 and have an increased H3K27 methylation ( 33 ).…”

Fibulin-2: A Novel Biomarker for Differentiating Grade II from Grade I Meningiomas

“…Meningioma specimens were collected during planned tumour resections; these are surplus samples destined for the incinerator following histological analysis for diagnosis and processed using techniques previously described [ 33 , 45 ].…”

“…The tumours were then minced into small pieces and disaggregated in digestion media (Dulbecco’s Modified Eagle Medium, DMEM supplemented with 10% FBS, 100 U/mL Penicillin, 100 U/mL Streptomycin and 20 units/mL of Type III collagenase (Worthington Biochemical Corp, Lakewood Township, NJ, USA)) overnight at 37 °C. Following digestion, the cells were pelleted at 1500 rpm for 5 min, the supernatant was removed and the pellet re-suspended in the complete medium and seeded in the appropriate tissue culture dish [ 45 ].…”

“…Protein extraction, quantification and cell culture Western blots were performed as previously described [ 12 , 33 , 45 , 46 , 47 , 48 ]. Antibodies used are listed in Supplementary data S3 .…”

“…Total RNA was extracted from 62 frozen tissues and cells using the Qiazol reagent (Qiagen UK), according to the manufacturer’s protocol. The ThermoFisher Scientific Nanodrop 2000 Spectrophotometer (Waltham, MA, USA) was used to assess the quality and concentration of the RNA [ 33 , 45 ].…”