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

Bechet D, Gielen GG, Korshunov A, Pfister SM, Rousso C, Faury D, Fiset PO, Benlimane N, Lewis PW, Lu C, David Allis C, Kieran MW, Ligon KL, Pietsch T, Ellezam B, Albrecht S, Jabado N - Acta Neuropathol. (2014)

Bottom Line: 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.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.These results demonstrate that mutant H3K27M can be specifically identified with high specificity and sensitivity using an H3K27M antibody and immunohistochemistry.

View Article: PubMed Central - PubMed

Affiliation: Departments of Experimental Medicine and of Human Genetics, McGill University, 4060 Ste Catherine West, PT239, Montreal, QC, H3Z2Z3, Canada.

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.

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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)
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Fig1: 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)

Mentions: We then assessed H3K27M expression by IHC on the 143 genotyped HGA (118 GBM, 14 GBM recurrences, 11 AA, Table 1). Results were compared to H3K27me3 staining on consecutive sections and to genomic data. Anti-H3K27 M antibody showed a strong nuclear staining of most tumor cells (>80 %) in all 48 samples with the H3K27M genotype (Fig. 1; Tables 1, 2). None of the H3K27M wild-type samples showed nuclear staining (Table 2; Fig. 1; Supplementary Fig. 2). Importantly, the antibody was able to recognize both H3.3 and H3.1 K27M mutant tumors (Fig. 2), as expected given that the peptide it was raised against is identical in both mutant H3 variants. The anti-H3K27M antibody did not stain any of the H3.3G34R/V HGA included in this dataset (n = 7). It did not recognize endothelial cells or other vascular structures within H3K27M mutant HGA which still expressed H3K27me3 (Fig. 1; Supplementary Fig. 2).Fig. 1


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

Bechet D, Gielen GG, Korshunov A, Pfister SM, Rousso C, Faury D, Fiset PO, Benlimane N, Lewis PW, Lu C, David Allis C, Kieran MW, Ligon KL, Pietsch T, Ellezam B, Albrecht S, Jabado N - Acta Neuropathol. (2014)

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)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig1: 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)
Mentions: We then assessed H3K27M expression by IHC on the 143 genotyped HGA (118 GBM, 14 GBM recurrences, 11 AA, Table 1). Results were compared to H3K27me3 staining on consecutive sections and to genomic data. Anti-H3K27 M antibody showed a strong nuclear staining of most tumor cells (>80 %) in all 48 samples with the H3K27M genotype (Fig. 1; Tables 1, 2). None of the H3K27M wild-type samples showed nuclear staining (Table 2; Fig. 1; Supplementary Fig. 2). Importantly, the antibody was able to recognize both H3.3 and H3.1 K27M mutant tumors (Fig. 2), as expected given that the peptide it was raised against is identical in both mutant H3 variants. The anti-H3K27M antibody did not stain any of the H3.3G34R/V HGA included in this dataset (n = 7). It did not recognize endothelial cells or other vascular structures within H3K27M mutant HGA which still expressed H3K27me3 (Fig. 1; Supplementary Fig. 2).Fig. 1

Bottom Line: 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.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.These results demonstrate that mutant H3K27M can be specifically identified with high specificity and sensitivity using an H3K27M antibody and immunohistochemistry.

View Article: PubMed Central - PubMed

Affiliation: Departments of Experimental Medicine and of Human Genetics, McGill University, 4060 Ste Catherine West, PT239, Montreal, QC, H3Z2Z3, Canada.

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.

Show MeSH
Related in: MedlinePlus