Four methods to analyze H3K27M mutation in diffuse midline gliomas

The histone H3 K27M mutation has been frequently reported in the majority of diffuse midline gliomas, which is considered as a prognostic and predictive biomarker. A number of different methods and platforms including pyrosequencing (PSQ), sanger sequencing, immunohistochemistry (IHC), Mass array and NGS (Next Generation Sequencing) have been used to detect H3K27M mutation in diffuse midline gliomas. However, controversy remains about the most appropriate method to use for analyzing H3K27M status. The H3K27 M mutation status of a total of 50 diffuse midline gliomas was examined using PSQ, sanger sequencing, IHC and Mass array in parallel. Using PSQ as a recommended standard method, the sensitivity, specificity and correlation with the other assays were calculated. Among 50 diffuse midline glioma cases, the H3K27M mutation was positive in 64 %, 66 %, 62 % and 62 % of the cases by PSQ, IHC, sanger sequencing and mass array, respectively. The sensitivity and specificity of IHC were 100 % and 94.4 %, respectively. The sensitivity and specificity of sanger sequencing and mass array were both 96.9 % and 100 %, respectively.This study demonstrated that IHC is an effective and rapid detection method for routine use in pathology laboratories for the identification of H3K27M mutation. A combination of IHC and sanger sequencing assays can provide 100 % sensitivity and specificity for the prediction of H3K27M status.     

Co‐occurrence of histone H3 K27M and BRAF V600E mutations in paediatric midline grade I ganglioglioma

Ganglioglioma (GG) is a grade I tumor characterized by alterations in the MAPK pathway, including BRAF V600E mutation. Recently, diffuse midline glioma with an H3 K27M mutation was added to the WHO 2016 classification as a new grade IV entity. As co‐occurrence of H3 K27M and BRAF V600E mutations has been reported in midline tumors and anaplastic GG, we searched for BRAF V600E and H3 K27M mutations in a series of 54 paediatric midline grade I GG (midline GG) to determine the frequency of double mutations and its relevance for prognosis. Twenty‐seven patients (50%) possessed the BRAF V600E mutation. The frequency of the co‐occurrence of H3F3A/BRAF mutations at diagnosis was 9.3%. No H3 K27M mutation was detected in the absence of the BRAF V600E mutation. Double‐immunostaining revealed that BRAF V600E and H3 K27M mutant proteins were present in both the glial and neuronal components. Immunopositivity for the BRAF V600E mutant protein correlated with BRAF mutation status as detected by massARRAY or digital droplet PCR. The median follow‐up of patients with double mutation was 4 years. One patient died of progressive disease 8 years after diagnosis, whereas the four other patients were all alive with stable disease at the last clinical follow‐up (at 9 months, 1 year and 7 years) without adjuvant therapy. We demonstrate in this first series of midline GGs that the H3 K27M mutation can occur in association with the BRAF V600E mutation in grade I glioneuronal tumors. Despite the presence of H3 K27M mutations, these cases should not be graded and treated as grade IV tumors because they have a better spontaneous outcome than classic diffuse midline H3 K27M‐mutant glioma. These data suggest that H3 K27M cannot be considered a specific hallmark of grade IV diffuse gliomas and highlight the importance of integrated histomolecular diagnosis in paediatric brain tumors.     

An H3F3A K27M‐mutation in a sonic hedgehog medulloblastoma

Medulloblastomas are malignant embryonal brain tumours that may harbour mutations in histone‐modifying genes, while mutations in histone genes have not been detected to date. We here describe the first SHH medulloblastoma with H3 K27M mutation. This may have diagnostic implications as H3 K27M mutations are the hallmark of diffuse midline gliomas, H3 K27M mutant, WHO grade IV. Medulloblastomas arise in midline structures and thus must not be mistaken for DMG when using an antibody detecting the H3 K27M mutation.     

Diffuse Midline Gliomas with Histone H3‐K27M Mutation: A Series of 47 Cases Assessing the Spectrum of Morphologic Variation and Associated Genetic Alterations

Somatic mutations of the H3F3A and HIST1H3B genes encoding the histone H3 variants, H3.3 and H3.1, were recently identified in high‐grade gliomas arising in the thalamus, pons and spinal cord of children and young adults. However, the complete range of patients and locations in which these tumors arise, as well as the morphologic spectrum and associated genetic alterations remain undefined. Here, we describe a series of 47 diffuse midline gliomas with histone H3‐K27M mutation. The 25 male and 22 female patients ranged in age from 2 to 65 years (median = 14). Tumors were centered not only in the pons, thalamus, and spinal cord, but also in the third ventricle, hypothalamus, pineal region and cerebellum. Patients with pontine tumors were younger (median = 7 years) than those with thalamic (median = 24 years) or spinal (median = 25 years) tumors. A wide morphologic spectrum was encountered including gliomas with giant cells, epithelioid and rhabdoid cells, primitive neuroectodermal tumor (PNET)‐like foci, neuropil‐like islands, pilomyxoid features, ependymal‐like areas, sarcomatous transformation, ganglionic differentiation and pleomorphic xanthoastrocytoma (PXA)‐like areas. In this series, histone H3‐K27M mutation was mutually exclusive with IDH1 mutation and EGFR amplification, rarely co‐occurred with BRAF‐V600E mutation, and was commonly associated with p53 overexpression, ATRX loss (except in pontine gliomas), and monosomy 10.     

Evaluation of Histone 3 Lysine 27 Trimethylation (H3K27me3) and Enhancer of Zest 2 (EZH2) in Pediatric Glial and Glioneuronal Tumors Shows Decreased H3K27me3 in H3F3A K27M Mutant Glioblastomas

H3F3A mutations are seen in ～30% of pediatric glioblastoma (GBMs) and involve either the lysine residue at position 27 (K27M) or glycine at position 34 (G34R/V). Sixteen genes encode histone H3, each variant differing in only a few amino acids. Therefore, how mutations in a single H3 gene contribute to carcinogenesis is unknown. H3F3A K27M mutations are predicted to alter methylation of H3K27. H3K27me3 is a repressive mark critical to stem cell maintenance and is mediated by EZH2, a member of the polycomb‐group (PcG) family. We evaluated H3K27me3 and EZH2 expression using immunohistochemistry in 76 pediatric brain tumors. H3K27me3 was lowered/absent in tumor cells but preserved in endothelial cells and infiltrating lymphocytes in six out of 20 GBMs. H3K27me3 showed strong immunoreactivity in all other tumor subtypes. Sequencing of GBMs showed H3F3A K27M mutations in all six cases with lowered/absent H3K27me3. EZH2 expression was high in GBMs, but absent/focal in other tumors. However, no significant differences in EZH2 expression were observed between H3F3A K27M mutant and wild type GBMs, suggesting that EZH2 mediated trimethylation of H3K27 is inhibited in GBM harboring K27M mutations. Our results indicate that H3F3A K27M mutant GBMs show decreased H3K27me3 that may be of both diagnostic and biological relevance.     

Alternative lengthening of telomeres,ATRX loss and H3‐K27M mutations in histologically defined pilocytic astrocytoma with anaplasia

Anaplasia may be identified in a subset of tumors with a presumed pilocytic astrocytoma (PA) component or piloid features, which may be associated with aggressive behavior, but the biologic basis of this change remains unclear. Fifty‐seven resections from 36 patients (23 M, 13 F, mean age 32 years, range 3–75) were included. A clinical diagnosis of NF1 was present in 8 (22%). Alternative lengthening of telomeres (ALT) was assessed by telomere‐specific FISH and/or CISH. A combination of immunohistochemistry, DNA sequencing and FISH were used to study BRAF, ATRX, CDKN2A/p16, mutant IDH1 p.R132H and H3‐K27M proteins. ALT was present in 25 (69%) cases and ATRX loss in 20 (57%), mostly in the expected association of ALT+/ATRX‐ (20/24, 83%) or ALT‐/ATRX+ (11/11, 100%). BRAF duplication was present in 8 (of 26) (31%). H3‐K27M was present in 5 of 32 (16%) cases, all with concurrent ATRX loss and ALT. ALT was also present in 9 (of 11) cases in the benign PA precursor, 7 of which also had ATRX loss in both the precursor and the anaplastic tumor. In a single pediatric case, ALT and ATRX loss developed in the anaplastic component only, and in another adult case, ALT was present in the PA‐A component only, but ATRX was not tested. Features associated with worse prognosis included subtotal resection, adult vs. pediatric, presence of a PA precursor preceding a diagnosis of anaplasia, necrosis, presence of ALT and ATRX expression loss. ALT and ATRX loss, as well as alterations involving the MAPK pathway, are frequent in PA with anaplasia at the time of development of anaplasia or in their precursors. Additionally, a small subset of PA with anaplasia have H3‐K27M mutations. These findings further support the concept that PA with anaplasia is a neoplasm with heterogeneous genetic features and alterations typical of both PA and diffuse gliomas.     

H3K27me3 and VEGF is associated with poor prognosis in patients with synovial sarcoma

Purpose

Previous studies have shown a correlation between the expression of H3K27me3 and pathological characteristics of malignant tumors. This study aimed to investigate the association of H3K27me3 and VEGF expression with clinical outcomes of synovial sarcoma patients.

Immunohistochemical evaluation of H3K27 trimethylation in malignant peripheral nerve sheath tumors

The histological definitive diagnosis of malignant peripheral nerve sheath tumor (MPNST) is quite difficult because the morphological features are not specific and no useful immunohistochemical marker has been identified. Loss-of-function mutations in EED or SUZ12, which encode the core subunit of polycomb repressive complex 2 (PRC2), were reported in MPNSTs, and the mutations were shown to cause inactivation of PRC2, leading to loss of trimethylation of histone H3 at lysine 27 (H3K27me3). Immunohistochemistry of H3K27me3 is expected to be a specific marker for MPNSTs. We evaluated immunohistochemical expression of H3K27me3 in MPNSTs with heterologous components and metachronous cases of MPNSTs. Among 145 MPNST samples, 50 (34.5%) showed complete loss of staining, and 45 (31.0%) showed partial loss of staining. Regarding the backgrounds of MPNSTs, 43 patients of neurofibromatosis type 1 (NF-1)-associated MPNST demonstrated 19 (44.2%) complete and 12 (27.9%) partial loss of H3K27me3. Among MPNSTs with heterologous component, almost all of MPNSTs with epithelioid differentiation (8/9 samples, 88.9%) retained H3K27me3, and malignant Triton tumors without epithelioid component lacked H3K27me3 at high rate (91.7%). Five of 20 metachronous MPNST cases showed significantly reduced expression of H3K27me3 between primary and later-occurring tumors, but in some cases increased expression of H3K27me3 in the clinical course (such as complete loss to partial loss) was observed. If the tumors are recurrent or metastatic, H3K27me3 expression should be reduced or at least maintained because loss of H3K27me3 is due to genetic mutation of EED or SUZ12. MPNSTs, especially those associated with NF-1, can occur in heterochronous and multiple patterns, and the identification of increased expression of H3K27me3 during a patient’s clinical course can be helpful for determining whether the tumors are heterochronous, multiple or not. As heterochronous and multiple tumors may show lower malignancy compared to recurrent or metastatic tumors, favorable prognosis may be expected when H3K27me3 expression is increased.     

Mallory-Denk bodies form when EZH2/H3K27me3 fails to methylate DNA in the nuclei of human and mice liver cells

EZH2/H3K27me3 and polycomb group complex (PcG) play a major role in regulating global gene expression including tumor suppressor genes. EZH2 is linked to cell cycle regulated EZH2 phosphorylation by CDK1, a mitotic kinase which increases in arrested mitosis compared to S phase. CDK1 phosphorylation of EZH2 accelerates the degradation of pEZH2. Phospho-EZH2 is subjected to ubiquitination. The half-like of pEZH2 is shorter when compared to total EZH2. In the present study, pEZH2 was found concentrated together with ubiquitin in the Mallory-Denk bodies (MDB) that were formed in hepatocytes in the livers of drug primed mice refed DDC and humans with alcoholic hepatitis or hepatocellular carcinoma. The cells that formed MDBs in the mice livers studied were associated with a growth advantage and a high proliferative index. However, the livers from patients with alcoholic hepatitis showed evidence of cell cycle arrest where PCNA, cyclin D1 and p27 positive nuclei were numerous but Ki-67 positive nuclei were scarce. It is concluded that MDB formation is linked to the cell cycle and global gene expression (i.e. loss of gene silencing) through its association with the regulation of the polycomb group PRC2/EZH2/H3K27me3 complex.     

H3K27me3 expression and methylation status in histological variants of malignant peripheral nerve sheath tumours

Diagnosing MPNST can be challenging, but genetic alterations recently identified in polycomb repressive complex 2 (PRC2) core component genes, EED and SUZ12, resulting in global loss of the histone 3 lysine 27 trimethylation (H3K27me3) epigenetic mark, represent drivers of malignancy and a valuable diagnostic tool. However, the reported loss of H3K27me3 expression ranges from 35% to 84%. We show that advances in molecular pathology now allow many MPNST mimics to be classified confidently. We confirm that MPNSTs harbouring mutations in PRC2 core components are associated with loss of H3K27me3 expression; whole-genome doubling was detected in 68%, and SSTR2 was amplified in 32% of MPNSTs. We demonstrate that loss of H3K27me3 expression occurs overall in 38% of MPNSTs, but is lost in 76% of histologically classical cases, whereas loss was detected in only 23% cases with heterologous elements and 14% where the diagnosis could not be provided on morphology alone. H3K27me3 loss is rarely seen in other high-grade sarcomas and was not found to be associated with an inferior outcome in MPNST. We show that DNA methylation profiling distinguishes MPNST from its histological mimics, was unrelated to anatomical site, and formed two main clusters, MeGroups 4 and 5. MeGroup 4 represents classical MPNSTs lacking H3K27me3 expression in the majority of cases, whereas MeGroup 5 comprises MPNSTs exhibiting non-classical histology and expressing H3K27me3 and cluster with undifferentiated sarcomas. The two MeGroups are distinguished by differentially methylated PRC2-associated genes, the majority of which are hypermethylated in the promoter regions in MeGroup 4, indicating that the PRC2 target genes are not expressed in these tumours. The methylation profiles of MPNSTs with retention of H3K27me3 in MeGroups 4 and 5 are independent of mutations in PRC2 core components and the driver(s) in these groups remain to be identified. Our results open new avenues of investigation. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.     

Phosphohistone‐H3 (PHH3) is prognostic relevant in Merkel cell carcinomas but Merkel cell polyomavirus is a more powerful prognostic factor than AJCC clinical stage,PHH3, Ki‐67 or mitotic indices

Merkel cell carcinomas (MCCs) associated with Merkel cell polyomavirus (MCPyV) have better prognosis than those without MCPyV. The relationship between mitotic index (MI) and MCC outcome has remained elusive because of the difficulty in differentiating mitotic cells from apoptotic ones. We evaluated the role of phosphohistone‐H3 (PHH3) (Ser10), a new mitotic count biomarker, in MCPyV‐positive or ‐negative MCC patients, and assessed its prognostic value in comparison to Ki‐67 labeling index or MI using hematoxylin and eosin (HE) staining. We compared the prognostic value of PHH3 mitotic index with that of MI by HE in 19 MCPyV‐positive and 9 MCPyV‐negative MCC patients. PHH3‐positive immunoreactivity was mostly observed in mitotic figures. Multivariate analysis significantly showed that MCPyV status (HR, 0.004; 95% CI 0.0003–0.058) and the American Joint Committee of Cancer (AJCC) stage (HR, 5.02; 95% CI 1.23–20.51) were observed as significantly independent prognostic factors for OS. PHH3‐positive cell counts/10 HPF was a slightly significant independent prognostic factor for OS (HR, 4.96; 95% CI 0.93–26.55). PHH3‐positive MI and MCPyV status in MCC patients are useful in prognostication, although MCPyV‐infection is a more powerful prognostic factor in MCCs than the AJCC scheme on proliferation or mitotic indices.     

Regional intestinal adaptations in Na+,K+‐ATPase in experimental colitis and the contrasting effects of interferon‐γ

Aims: This study evaluated Na⁺,K⁺‐ATPase activity and the abundance of α₁ subunit Na⁺,K⁺‐ATPase in experimental colitis and gathered evidence on the effects of interferon‐γ (IFN‐γ) on intestinal Na⁺,K⁺‐ATPase. Methods: Colitis was induced by the intrarectal administration of 2,4,6‐trinitrobenzene sulphonic acid (TNBS, 30 mg/250 μL). Na⁺,K⁺‐ATPase activity was determined as the difference between total and ouabain‐insensitive ATPase. The abundance of Na⁺,K⁺‐ATPase was analysed by immunoblotting. Results: Na⁺,K⁺‐ATPase activity was markedly reduced in the proximal colonic mucosa of TNBS‐treated rats, whereas upstream in the terminal ileal mucosa a marked increase in sodium pump activity was observed. At the jejunal level no significant changes in Na⁺,K⁺‐ATPase activity were observed between TNBS‐treated rats and corresponding controls (ethanol‐treated rats). No changes were observed in the abundance of α₁ subunit Na⁺,K⁺‐ATPase in the proximal colon, terminal ileum and jejunum. The administration of IFN‐γ (50 000 U) 48 h before sacrifice reduced both Na⁺,K⁺‐ATPase activity and the abundance of α₁ subunit Na⁺,K⁺‐ATPase in the proximal colon. Dexamethasone prevented colonic inflammation and decreases in proximal colonic Na⁺,K⁺‐ATPase activity in TNBS‐treated rats, but did not affect the INF‐γ‐induced decrease in colonic Na⁺,K⁺‐ATPase activity. Conclusions: The increase in ileal Na⁺,K⁺‐ATPase activity upstream to the lesioned colonic mucosa, where Na⁺,K⁺‐ATPase activity was markedly reduced, might indicate a compensatory process to counteract the decrease in water and electrolyte absorption at the colonic level. This decrease in colonic Na⁺,K⁺‐ATPase activity is likely not related to INF‐γ‐induced downregulation of Na⁺,K⁺‐ATPase.