Evaluation of a novel antibody to define histone 3.3 G34R mutant brain tumours

Abstract

Missense somatic mutations affecting histone H3.1 and H3.3 proteins are now accepted as the hallmark of paediatric diffuse intrinsic pontine gliomas (DIPG), non-brain stem paediatric high grade gliomas (pHGG) as well as a subset of adult glioblastoma multiforme (GBM). Different mutations give rise to one of three amino acid substitutions at two critical positions within the histone tails, K27M, G34R/V. Several studies have highlighted gene expression and epigenetic changes associated with histone H3 mutations; however their precise roles in tumourigenesis remain incompletely understood. Determining how such amino acid substitutions in a protein affect its properties can be challenging because of difficulties in detecting and tracking mutant proteins within cells and tissues. Here we describe a strategy for the generation of antibodies to discriminate G34R and G34V mutant histone H3 proteins from their wild-type counterparts. Antibodies were validated by western blotting and immunocytochemistry, using recombinant H3.3 proteins and paediatric GBM cell lines. The H3-G34R antibody demonstrated a high degree of selectivity towards its target sequence. Accordingly, immunostaining on a cohort of 22 formalin-fixed paraffin embedded tumours with a previously known H3.3 G34R mutation status, detected successfully the corresponding mutant protein in 11/11 G34R cases. Since there was a high concordance between genotype and immunohistochemical analysis of G34R mutant tumour samples, we analysed a series of tissue microarrays (TMAs) to assess the specificity of the antibody in a range of paediatric brain tumours, and noted immunoreactivity in 2/634 cases. Importantly, we describe the generation and validation of highly specific antibodies for G34 mutations. Overall our work adds to an extremely valuable portfolio of antibodies, not only for histopathologic detection of tumour-associated mutant histone sequences, but also facilitating the study of spatial/anatomical aspects of tumour formation and the identification of downstream targets and pathways in malignant glioma progression.

Molecular analysis of pediatric CNS-PNET revealed nosologic heterogeneity and potent diagnostic markers for CNS neuroblastoma with FOXR2-activation

“…However this technology requires resources not accessible on a global scale. Likewise, GBM_ G34 and EPN_RELA are readily diagnosed by methylation analysis, however, both entities can be identified recently by targeting the H3.3 G34 mutation or by employing p65-RelA antibodies [9,21]. In contrast, the unequivocal recognition of GBM_MYCN with non-molecular methods still poses challenges.…”

Radiological and Immunostaining Characteristics of H3.3 G34R-Mutant Glioma: A Report of 3 Cases and Review of the Literature

“…The use of immunostaining against IDH1-R132H antibody is widely accepted and applied [13]. Haque et al [14] discovered that immunostaining for H3.3 G34R/V-mutant in glioma is also highly concordant with the genotype analysis. Our cases confirmed that the use of immunohistochemistry staining to detect H3.3 G34R-mutations is more efficient, compared Positive cutoff values of ATRX and p53 were 10% nuclear staining.…”

Pediatric Glial Tumors

“…For hemispheric tumors, immunostains for H3.3 p.G34R and H3.3 p.G34V are available; however sequencing is more specific. 54…”