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

Acta Neuropathologica Communications

Lisa Storer¹  Ruman Rahman¹  Farhana Haque¹  Richard G Grundy¹  Jacques Grill²  Julien Puntonet³  Pascale Varlet³  Chris Jones⁴  Angel M Carcaboso⁵  Aikaterini Bountali⁶  Robert Layfield⁷ 

[1] Children’s Brain Tumour Research Centre (CBTRC), School of Medicine, Queen’s Medical Centre, University of Nottingham;Departement de Cancerologie de l’Enfant et de l’Adolescent et Unité Mixte de Recherche 8203 du Centre National de la Recherche Scientifique;Department of Neuropathology, Sainte-Anne Hospital;Divisions of Molecular Pathology and Cancer Therapeutics, The Institute of Cancer Research;Institut de Recerca Sant Joan de Deu;School of Life Sciences, Faculty of Natural Sciences, Keele University;School of Life Sciences, Queen’s Medical Centre, University of Nottingham;

关键词: Histone mutations;    H3.3;    H3.1;    DIPG;    pHGG;    Brain tumour;   

DOI  :  10.1186/s40478-017-0449-1

来源: DOAJ

【 摘 要 】

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.

【 授权许可】

Unknown