Specific detection of methionine 27 mutation in histone 3 variants (H3K27M) in fixed tissue from high-grade astrocytomas

Abstract

Studies in pediatric high-grade astrocytomas (HGA) by our group and others have uncovered recurrent somatic mutations affecting highly conserved residues in histone 3 (H3) variants. One of these mutations leads to analogous p.Lys27Met (K27M) mutations in both H3.3 and H3.1 variants, is associated with rapid fatal outcome, and occurs specifically in HGA of the midline in children and young adults. This includes diffuse intrinsic pontine gliomas (80 %) and thalamic or spinal HGA (>90 %), which are surgically challenging locations with often limited tumor material available and critical need for specific histopathological markers. Here, we analyzed formalin-fixed paraffin-embedded tissues from 143 pediatric HGA and 297 other primary brain tumors or normal brain. Immunohistochemical staining for H3K27M was compared to tumor genotype, and also compared to H3 tri-methylated lysine 27 (H3K27me3) staining, previously shown to be drastically decreased in samples carrying this mutation. There was a 100 % concordance between genotype and immunohistochemical analysis of H3K27M in tumor samples. Mutant H3K27M was expressed in the majority of tumor cells, indicating limited intra-tumor heterogeneity for this specific mutation within the limits of our dataset. Both H3.1 and H3.3K27M mutants were recognized by this antibody while non-neoplastic elements, such as endothelial and vascular smooth muscle cells or lymphocytes, did not stain. H3K27me3 immunoreactivity was largely mutually exclusive with H3K27M positivity. These results demonstrate that mutant H3K27M can be specifically identified with high specificity and sensitivity using an H3K27M antibody and immunohistochemistry. Use of this antibody in the clinical setting will prove very useful for diagnosis, especially in the context of small biopsies in challenging midline tumors and will help orient care in the context of the extremely poor prognosis associated with this mutation.

Fig. 1

Immunohistochemical (IHC) staining of pediatric high-grade astrocytomas (HGA) using the anti-H3K27M antibody correlates with tumor genotype and decreased H3K27Me3 in tumors. Representative IHC of pediatric HGA using anti-H3K27M (a, c, e, g) or anti-H3K27me3 (b, d, f, h) antibodies and counterstained with hematoxylin. H3K27M shows strong nuclear positivity in tumor cells, but no staining in the nuclei of endothelial and smooth muscle cells in blood vessels in K27M mutant tumors (a, c). Tumors wild type for H3K27 show no nuclear staining with the anti-H3K27M antibody (e, g). Corresponding H3K27me3 staining on the same samples shows global decrease of the expression of this histone mark in H3K27M mutant tumors (b, d) compared to tumors wild type for this mutation (f, h). Notably, positivity for H3K27me3 was mainly seen in tumor vessels (b, d) even though a degree of intra-tumor staining was also seen in H3K27M mutant samples (d)

Fig. 2

Immunohistochemical (IHC) staining of H3.1K27M and H3.3K27M mutant high-grade astrocytomas (HGA) shows similar results using the anti-H3K27M and anti-H3K27me3 antibodies. Representative sections from HGAs (WHO Grade III) carrying H3.3K27M (a, b) and H3.1K27M (c, d, e, f) immunostained with anti-H3K27M (a, c, e) and anti-H3K27me3 (b, d, f) antibodies. Two areas from the same H3.1K27M WHO Grade III DIPG sample are shown to further illustrate that the K27M mutation is also present in seemingly lower grade tumor sections (c)

Fig. 3

H3K27M staining and H3K27me3/CD45 co-immunostaining on consecutive slides of an H3K27M mutant high-grade astrocytoma. a anti-H3K27M staining (nuclear); b co-immunostaining using a CD45 antibody (cytoplasmic, purple) and an H3K27me3 antibody (nuclear, dark brown); results indicate that only a proportion of cells positive for H3K27me3 staining in H3K27M mutant high-grade astrocytomas are positive for CD45 staining and represent a lymphocytic infiltrate (light blue arrows). Endothelial and smooth muscle vascular cells show uptake of the H3K27me3 antibody alone (asterisk) while few cells within the tumor remain positive for H3K27me3 (black arrow)