Characterisation of Expression the Arginine Pathway Enzymes in Childhood Brain Tumours to Determine Susceptibility to Therapeutic Arginine Depletion

Abstract

Despite significant improvements in treatment and survival in paediatric cancers, outcomes for children with brain tumours remain poor. Novel therapeutic approaches are needed to improve survival and quality of survival. Extracellular arginine dependency (auxotrophy) has been recognised in several tumours as a potential therapeutic target. This dependency is due to the inability of cancer cells to recycle or synthesise intracellular arginine through the urea cycle pathway compared to normal cells. Whilst adult glioblastoma exhibits this dependency, the expression of the arginine pathway enzymes has not been delineated in paediatric brain tumours. We used immunohistochemical (IHC) methods to stain for arginine pathway enzymes in paediatric high-grade glioma (pHGG), low-grade glioma (pLGG), ependymoma (EPN), and medulloblastoma (MB) tumour tissue microarrays (TMAs). The antibodies detected protein expression of the metaboliser arginase (Arg1 and Arg2); recycling enzymes ornithine transcarbamoylase (OTC), argininosuccinate synthetase (ASS1), and argininosuccinate lyase (ASL); and the transporter SLC7A1. Deficiency of OTC, ASS1, and ASL was seen in 87.5%, 94%, and 79% of pHGG samples, respectively, consistent with an auxotrophic signature. Similar result was obtained in pLGG with 96%, 93%, and 91% of tumours being deficient in ASL, ASS1, and OTC, respectively. 79%, 88%, and 85% of MB cases were ASL, ASS1, and OTC deficient whilst ASL and OTC were deficient in 57% and 91% of EPN samples. All tumour types highly expressed SLC7A1 and Arginase, with Arg2 being the main isoform, demonstrating that they could transport and utilise arginine. Our results show that pHGG, pLGG, EPN, and MB demonstrate arginine auxotrophy based on protein expression and are likely to be susceptible to arginine depletion. Pegylated arginase (BCT-100) is currently in phase I/II trials in relapsed pHGG. Our results suggest that therapeutic arginine depletion may also be useful in other tumour types and IHC analysis of patient tumour samples could help identify patients likely to benefit from this treatment.

Figure 1

Scoring criteria for IHC. (a) Cores were scored as having negative (0%), very low (1-5%), low (5-20%), moderate (20-50%), or high (>50%) expression. (b) Squares represent areas where staining was assessed in each core. (c) Example of positive control tissue (liver).

Figure 2

Expression of arginine pathway enzymes in pHGG expressed as a percentage normalised to 100% of the total number of cases scored per antibody. Tumours were classed as being deficient if the expression level of an individual pathway enzyme was 20% or lower. High expression was defined as >50% antibody staining (see supplementary table 1 for number of samples analysed per antibody in the pHGG cohort).

Figure 3

Expression of arginine pathway enzymes in LGG expressed as a percentage normalised to 100% of the total number of cases scored per antibody. Tumours were classed as being deficient if the expression level of an individual pathway enzyme was 20% or lower. High expression was defined as >50% antibody staining (see supplementary table 2 for number of samples analysed per antibody in the LGG cohort).

Figure 4

Expression of arginine pathway enzymes in MB expressed as a percentage normalised to 100% of the total number of cases scored per antibody. Tumours were classed as being deficient if the expression level of an individual pathway enzyme was 20% or lower. High expression was defined as >50% antibody staining (see supplementary table 3 for number of samples analysed per antibody in the MB cohort).

Figure 5

Expression of arginine pathway enzymes in EPN expressed as a percentage normalised to 100% of the total number of cases scored per antibody. Tumours were classed as being deficient if the expression level of an individual pathway enzyme was 20% or lower. High expression was defined as >50% antibody staining (see supplementary table 4 for number of samples analysed per antibody in the EPN cohort).