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

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.     

Case series of diffuse extraneural metastasis in H3F3A mutant high-grade gliomas: Clinical,molecular phenotype and literature review

H3K27M and H3.3G34R/V mutations have been identified in pediatric high-grade gliomas (pHGG), though extraneural metastases are rarely reported and poorly characterized. Three pHGG patients from two institutions were identified with extraneural metastasis, harboring histone mutations. Their clinical, imaging and molecular characteristics are reported here. A 17-year old female presented with supratentorial H3.3G34R-mutant glioma with metastatic osseous lesions in the spine, pelvis, bone marrow, pleural effusion and soft tissue of pelvis. Bone marrow biopsy and soft tissue of pelvis biopsy showed neoplastic cells positive for P53. A 20-year old female was diagnosed with H3F3A H3K27M–mutant thalamic glioma. She developed diffuse sclerotic osseous lesions. Biopsy of an osseous lesion was non-diagnostic. A 17-year old female presented with a H3F3A H3K27M-mutant diffuse midline glioma with diffuse spinal cord metastasis. She further developed multifocal chest lymphadenopathy, pleural effusions, and a soft tissue mass in the abdominal wall. The latter was positive for H3K27M mutation. We present the first case series of pHGG with H3F3A mutation and diffuse extraneural dissemination, describing their clinical and molecular profile.     

H3K27M-mutant diffuse midline glioma presenting as synchronous lesions involving pineal and suprasellar region: A case report and literature review

IntroductionThe differential diagnoses for multifocal lesions with pineal and suprasellar involvement in a young adult include germ cell tumour and intracranial metastasis. Other differentials include atypical teratoid/rhabdoid tumour and pineoblastoma. We present the first known case of multicentric H3K27M mutant diffuse midline glioma, which is typically defined by its diffuse nature, midline location, and H3K27M mutation.Case reportA young Chinese female presented subacutely with giddiness, right abducens nerve palsy and unsteady gait. Magnetic resonance imaging (MRI) of the brain with contrast revealed a moderately sized pineal region tumour, extending into the third ventricle, associated with hydrocephalus. There were two other synchronous lesions noted in the suprasellar and left occipital region. Serum and cerebrospinal fluid tumour markers, along with a computed tomography scan of her thorax and abdomen and were unremarkable. She underwent an endoscopic third ventriculostomy and biopsy of pineal and suprasellar lesions. Histology demonstrated a poor prognosis variant multifocal glioblastoma multiforme that was IDH wildtype, H3K27M positive, and MGMT unmethylated. MRI of the whole spine did not reveal any drop metastasis. The patient subsequently underwent adjuvant chemotherapy and radiotherapy after she was deemed to be unsuitable for surgical resection.ConclusionAlthough rare, multicentric H3K27M mutant diffuse midline glioma should be included in the list of differential diagnoses for multifocal enhancing lesions with involvement of the pineal and suprasellar regions, especially if the lesions demonstrate imaging features atypical for more common diagnosis such as germ cell tumours.     

A case of diffuse midline glioma,H3 K27M mutant mimicking a hemispheric malignant glioma in an elderly patient

Diffuse midline glioma, H3 K27M mutant arises from midline structures of the central nervous system and predominately affects pediatric patients. However, this disease entity was only recently established, and the clinical phenotypic spectrum remains largely unclear. We herein report a rare case of diffuse midline glioma, H3 K27M mutant with an unusual distribution in an elderly woman who presented with a diffuse glioma that invaded both sides of the thalami, and left hippocampus and frontoparietal lobes, thus mimicking a hemispheric malignant glioma. A biopsy of the lobular lesion led to a molecular diagnostic confirmation of diffuse midline glioma, H3 K27M mutant. The patient received concurrent bevacizumab and temozolomide therapy with radiation therapy and survived for 30 months. This case highlights the possibility that a glioma with cerebral hemispheric spread in an elderly patient may harbor the H3 K27M mutation.     

Analysis of PD‐L1 expression and T cell infiltration in different molecular subgroups of diffuse midline gliomas

Diffuse midline gliomas (DMGs) are rare and devastating tumors with limited therapeutic options. Programmed death‐ligand 1 (PD‐L1) expression represents a potential predictive biomarker for immunotherapy. One hundred and twenty‐six DMGs (89 adult and 37 pediatric) were assessed for immune profile (PD‐L1, cluster of differentiation (CD3, CD8) and genetic markers (mutation in 27th amino acid of histone H3 (H3K27M), alpha thalassemia/mental retardation syndrome X‐linked (ATRX), isocitrate dehydrogenase 1 (IDH1), p53) by immunohistochemistry. Sanger sequencing was done for IDH1 and H3K27M. The thalamus was the commonest site. Four molecular subgroups of DMGs were identified. H3K27M mutation was more frequent in children (P = 0.0001). The difference in median overall survival (OS) was not significant in any of the four molecular subgroups (P > 0.05). PD‐L1 expression was significantly higher in H3K27M/IDH1 double‐negative adult glioblastomas (GBMs) (P = 0.002). Strong PD‐L1 expression was more frequent in grade IV tumors and thalamic location, although the difference was not significant (P = 0.14 and P = 0.19 respectively). Positive PD‐L1 expression was significantly associated with high tumor‐infiltrating lymphocytes count (P < 0.05). There was no significant difference in median OS in PD‐L1‐positive versus negative cases among four genetic subgroups (P > 0.05). On univariate analysis, there was no direct correlation of PD‐L1 with any genetic alteration, except H3K27M mutation (P = 0.01). CD3 infiltration was similar in both adults and pediatric ages (84.3% and 78.4%, respectively) while CD8 expression was significantly greater in adults compared to children (74.1% vs 37.8%, P = 0.0001). This is the first comprehensive analysis highlighting molecular and immune profiles of DMGs. Despite molecular and clinicopathological diversity, overall survival in DMGs remains dismal. Multicentric studies with larger numbers of cases should be undertaken for stratifying DMGs according to their age, immune and molecular profiles, to develop effective immunotherapies.     

p.N345K mutation in TARDBP in a patient with familial amyotrophic lateral sclerosis: An autopsy case

We report the neuropathology of a patient with a family history of amyotrophic lateral sclerosis (ALS) and a p.N345K mutation in the transactivation response DNA‐binding protein 43 kDa (TDP‐43) gene (TARDBP). A 62‐year‐old man had bulbar palsy with progressive weakness in the extremities. Neurological examination revealed evident upper motor neuron signs and lower motor neuron involvement corroborated by needle electromyography. The patient was diagnosed as having probable ALS according to the revised El Escorial diagnostic criteria and was eventually diagnosed with familial ALS. At 65 years of age, respiratory failure became critical, and artificial ventilation was initiated. At 70 years of age, the patient died from a urinary tract infection. Histopathological investigation showed Bunina bodies in the remaining motor neurons and anterolateral funicular myelin pallor in the spinal cord. TDP‐43‐positive cytoplasmic inclusions were quite rare in the spinal cord motor neurons, being predominantly present in the glial cells (especially astrocytes) of the spinal cord anterior horn. Although the reason for the preferential vulnerability of spinal glial cells to TARDBP mutations remains unclear, our findings indicate that TARDBP p.N345K mutation could have an influence on the topography of TDP‐43 aggregation.     

伴H3 K27M突变的儿童弥漫性中线胶质瘤的临床特点、治疗及预后

目的探讨伴H3 K27M突变的儿童弥漫性中线胶质瘤(DMG)的临床特点、治疗及预后。方法回顾性分析2017年7月至2020年3月首都医科大学附属北京儿童医院神经外科收治的10例DM G伴H3 K27M突变患儿的临床资料。10例患儿中,病变位于丘脑4例,脑干4例,脊髓2例,其中行开颅肿瘤切除术7例,脊髓肿瘤切除术2例,颅内病变活组织检查术1例4例术后行放疗联合化疗,1例行单纯放疗,5例未行放疗或化疗以肿瘤切除率>90%为近全切除,50%~90%为部分切除。术后随访至2020年7月或患儿死亡,随访患儿肿瘤有无进展、生存时间以及死亡原因。结果10例患儿的中位发病年龄为8.9(4.9~9.9)岁,颅内占位临床表现为头痛、恶心、呕吐、肢体无力、癫痫发作、意识障碍及脑神经麻痹;脊髓占位临床表现为进行性肢体无力。肿瘤近全切除4例,部分切除5例,活组织检查术1例。患儿术后症状改善6例,无改变4例;无一例出现中枢神经系统感染;1例在术后放疗期间出现脑积水10例患儿的肿瘤病理学结果均为DMG伴H3 K27M突变(世界卫生组织肿瘤分级Ⅳ级);免疫组织化学检测结果显示,少突胶质细胞转录因子2阳性比例为9/9,胶质纤维酸性蛋白阳性比例为10/10,突变型异柠檬酸脱氢酶1阳性比例为0/9,Ki-67≥40%的比例为8/10。10例患儿术后均得到有效随访;随访时间为1~23个月至末次随访,2例患儿存活,8例患儿因肿瘤进展死亡,生存时间为1~23个月结论初步观察发现,伴H3 K27M突变的儿童DMG临床表现多样,可选择手术、术后放疗和(或)化疗治疗,患儿的生存预后差。     

Molecular and clinical characterization of H3 K27M-mutant “non-midline” glioblastoma: A case report and literature review

The WHO classification of tumors of the CNS in 2016 defined “diffuse midline glioma, H3 K27M-mutant” as a new tumor entity locating in the CNS midline. However, the H3 K27M-mutation in “non-midline” glioblastoma are rare and their characteristics have been rarely reported. A 16-year-old girl presented a hyper-intense lesion at her left temporal stem on T2WI, FLAIR and DWI. Biopsy was performed and molecular pathological diagnosis was glioblastoma with H3 K27M-mutant. Accordingly, the possibility of H3 K27M-mutant should be examined not only for diffuse glioma without IDH mutation that develops at a midline location, but also in non-midline locations.     

A comprehensive analysis identifies BRAF hotspot mutations associated with gliomas with peculiar epithelial morphology

Brain tumors harbor various BRAF alterations, the vast majority of which are the BRAF kinase‐activating V600E mutation. BRAF mutations are most frequently detected in certain subtypes of low‐grade glioma, such as pilocytic astrocytoma (PA), pleomorphic xanthoastrocytoma (PXA), ganglioglioma (GG) and dysembryoplastic neuroepithelial tumor (DNT). However, it is unclear whether gliomas harboring BRAF mutations can be invariably regarded as these glioma subtypes or their derivatives. To address this question, we analyzed 274 gliomas in our institutional case series. We performed high‐resolution melting analyses and subsequent direct Sanger sequencing on DNA isolated from snap‐frozen tumor tissues. As expected, BRAF mutations were detected in the aforementioned low‐grade gliomas: in 4/27 PAs, 2/3 PXAs, 4/8 GGs, and 1/6 DNTs. In addition to these gliomas, 1/2 astroblastomas (ABs) and 2/122 glioblastomas (GBs) harbored BRAF mutations. Pathological investigation of the two GBs revealed that one was a GB displaying epithelial features that presumably arose from a precedent GG, whereas the other GB, which harbored a rare G596 A mutation, showed marked epithelial features, including astroblastic rosettes. Our results indicate that in addition to being present in established BRAF‐associated gliomas, BRAF mutations might be associated with epithelial features in high‐grade gliomas, including sheet‐like arrangement of polygonal tumor cells with a plump cytoplasm and astroblastic rosettes, and thus could potentially serve as a genetic marker for these features.     

A sensitive and specific histopathologic prognostic marker for H3F3A K27M mutant pediatric glioblastomas

Pediatric glioblastomas (GBM) are highly aggressive and lethal tumors. Recent sequencing studies have shown that ~30 % of pediatric GBM and ~80 % of diffuse intrinsic pontine gliomas show K27M mutations in the H3F3A gene, a variant encoding histone H3.3. H3F3A K27M mutations lead to global reduction in H3K27me3. Our goal was to develop biomarkers for the histopathologic detection of these tumors. Therefore, we evaluated the utility of measuring H3K27me3 global reduction as a histopathologic and prognostic biomarker and tested an antibody directed specifically against the H3.3 K27M mutation in 290 samples. The study cohort included 203 pediatric (including 38 pediatric high-grade astrocytomas) and 38 adult brain tumors of various subtypes and grades and 49 non-neoplastic reactive brain tissues. Detection of H3.3 K27M by immunohistochemistry showed 100 % sensitivity and specificity and was superior to global reduction in H3K27me3 as a biomarker in diagnosing H3F3A K27M mutations. Moreover, cases that stained positive for H3.3 K27M showed a significantly poor prognosis compared to corresponding negative tumors. These results suggest that immunohistochemical detection of H3.3 K27M is a sensitive and specific surrogate for the H3F3A K27M mutation and defines a prognostically poor subset of pediatric GBM.     

Cerebellar high‐grade astrocytoma with IDH mutations in the elderly: A report of two cases

Cerebellar high‐grade gliomas are rare, and likely to affect younger patients compared with those of cerebral origin. Recent genetic analyses have revealed that isocitrate dehydrogenase (IDH) 1/2 mutations are rare in infratentorial gliomas. In this paper, we report two elderly cases of IDH‐mutated cerebellar high‐grade glioma with unusual histological features and uncommon patient ages. One case was an 83‐year‐old man, whose tumor was predominantly composed of densely packed round‐to‐polygonal epithelioid cells. The other was a 75‐year‐old woman's high‐grade astrocytoma characterized by cord‐like structures and the perivascular papillary arrangements with varying amounts of myxoid matrix. The former harbored IDH1 R132H mutation, whereas the latter had IDH2 R172K mutation. According to our literature review, eight cases of IDH‐mutated infratentorial gliomas including the present cases have been reported, and four had mutations other than IDH1 R132H. Moreover, we herein report the first elderly case of IDH2‐mutation. Although the number is limited, IDH‐mutant infratentorial diffuse gliomas may have clinical, histological and genetic features different from supratentorial cases.     

Radiotherapy and radio-sensitization in H3K27M-mutated diffuse midline gliomas

Background

H3^K27M mutated diffuse midline gliomas (DMGs) are extremely aggressive and the leading cause of cancer-related deaths in pediatric brain tumors with 5-year survival <1%. Radiotherapy is the only established adjuvant treatment of H3^K27M DMGs; however, the radio-resistance is commonly observed.

Methods

We summarized current understandings of the molecular responses of H3^K27M DMGs to radiotherapy and provide crucial insights into current advances in radiosensitivity enhancement.

Results

Ionizing radiation (IR) can mainly inhibit tumor cell growth by inducing DNA damage regulated by the cell cycle checkpoints and DNA damage repair (DDR) system. In H3K27M DMGs, the aberrant genetic and epigenetic changes, stemness genotype, and epithelial-mesenchymal transition (EMT) disrupt the cell cycle checkpoints and DDR system by altering the associated regulatory signaling pathways, which leads to the development of radio-resistance.

Conclusions

The advances in mechanisms of radio-resistance in H3^K27M DMGs promote the potential targets to enhance the sensitivity to radiotherapy.     

Glial fibrillary acidic protein mutations in adult‐onset Alexander disease: clinical features observed in 12 Japanese patients

Yoshida T, Sasayama H, Mizuta I, Okamoto Y, Yoshida M, Riku Y, Hayashi Y, Yonezu T, Takata Y, Ohnari K, Okuda S, Aiba I, Nakagawa M. Glial fibrillary acidic protein mutations in adult‐onset Alexander disease: clinical features observed in 12 Japanese patients.
Acta Neurol Scand: 2011: 124: 104–108.
© 2010 John Wiley & Sons A/S. Objective – To clarify the clinical manifestations of adult‐onset Alexander disease (AOAD) in Japanese patients with glial fibrillary acidic protein (GFAP) gene mutations. Methods and materials – Twelve patients of AOAD with GFAP mutations detected in our centre were examined for neurological and magnetic resonance imaging (MRI) findings. Results – Major symptoms were pyramidal and bulbar signs. In addition, three patients presented abnormal behaviour and/or memory disturbance. Two of the three patients also had Parkinsonism and had been diagnosed with fronto‐temporal dementia or progressive supranuclear palsy until GFAP mutations were detected. Abnormalities of the medulla oblongata and cervical spinal cord were observed on MRI in all patients. Conclusions – Patients presenting with pyramidal and/or bulbar signs with abnormalities of the medulla oblongata and cervical spinal cord on MRI should be considered for GFAP analysis as this is the typical presentation of AOAD. Abnormal behaviour and cognitive disorders including deterioration of memory were rare symptoms but could be an obstacle to diagnosing Alexander disease.     

BRAF V600E mutation is a significant prognosticator of the tumour regrowth rate in brainstem gangliogliomas

BRAF V600E mutations are progression factors in paediatric low-grade gliomas. Furthermore, a high percentage of paediatric brainstem gangliogliomas have BRAF V600E mutations. However, their clinical significance, including possible connections between the biomarkers and ganglioglioma’s clinical features, especially a brainstem counterpart, is unclear. To identify potential molecular features predictive of brainstem ganglioglioma’s clinical outcomes, a retrospective cohort of 28 World Health Organization (WHO) grade I brainstem gangliogliomas was analysed for BRAF V600E, IDH1 R132H, and IDH2 R172K mutations, TERT C228T/C250T promoter mutation, H3F3A K27M mutation and MGMT methylation. The volume of tumours was calculated accurately by using 3D Slicer software. The clinical data of these patients were retrospectively analysed. In tumours with BRAF V600E mutations, the tumour regrowth rate was significantly faster than that of the wild type group (p = 0.001). Moreover, the BRAF V600E mutant group had shorter progression-free survival (PFS) compared with wild type (p = 0.012). On multivariate analysis, no factor was found to be an independent prognostic factor; however, tumours with faster regrowth rates had a strong trend towards an increased risk for shorter PFS (HR = 1.027, p = 0.056). No statistical analysis could be performed to evaluate factors affecting overall survival (OS). These data suggest that BRAF V600E can predict the regrowth rate of brainstem gangliogliomas after microsurgery, and a BRAF V600E-targeted therapeutic may be a promising early intervention measure for patients who harbour BRAF V600E mutation after microsurgery.     

Spinal cord anaplastic astrocytoma with BRAF V600E mutation: A case report and review of literature

A 17-year-old female complained of lower extremity pain that progressed to low back pain accompanied by paraparesis. Magnetic resonance imaging revealed a mass in the conus medullaris of the spinal cord at the thoracic spine 11–12 level. The patient underwent resection of the mass. The pathological diagnosis was anaplastic astrocytoma based on the densely proliferating astrocytic tumor cells without necrosis or microvascular proliferation. The patient received chemoradiotherapy with oral temozolomide and a total of 54 Gy of local irradiation, followed by 24 courses of temozolomide as maintenance chemotherapy. The patient survived for 8 years without tumor recurrence following the initial treatment. Genetic analysis of the tumor revealed a BRAF V600E mutation that has not yet been reported in spinal cord high-grade gliomas (HGGs). In recent years, the molecular therapy targeting the BRAF V600E mutation has been applied in clinical practice for several cancer types. Although the frequency in spinal cord HGGs is uncertain, it is necessary to investigate BRAF V600E mutation as a potential therapeutic target in the future.     

H3K27M mutation in adult cerebellar glioblastoma

A methionine substitution of lysine at residue 27 of histone H3 (H3K27M) mutation has become synonymous with malignant pediatric diffuse midline glioma (DMG), that occurs commonly in the brainstem. Therefore, recent reports that this same mutation occurs in malignant adult glioblastoma (GBM) located in the cerebellum are both unexpected and intriguing. The biological and clinical considerations of this novel finding are discussed.     

The clinicopathological and prognostic significance of TP53 alteration in K27M mutated gliomas: an individual-participant data meta-analysis

This study aimed to investigate the impact of TP53 alteration on survival and clinicopathological features of glioma patients with H3K27M mutations. An individual-participant-data (IPD) meta-analysis was performed to investigate the impact of TP53 alteration on survival and clinicopathological features of patients with H3K27M mutations. Three hundred thirty-one individual records from 12 eligible glioma studies involving the H3K27M mutation were finally included in our meta-analysis, and a pooled hazard ratio (HR) of 1.53 (95%CI, 1.10–2.11; P?=?0.01) indicated that TP53 alterations were associated with a shorter overall survival. The pooled odds ratios (ORs) indicated that TP53 alterations were significantly associated with the age at diagnosis ≥?7 years (OR?=?1.97, 95%CI?=?1.15–3.38, P =?0.01), the status of histone H3.3 mutations (OR?=?9.15, 95%CI?=?4.18–20.06, P < 0.00001), and high WHO grade histology (III?+?IV) (OR?=?2.70, 95%CI?=?1.33–5.48, P?=?0.006). However, no association was found between TP53 alterations and gender or tumor location. This IPD meta-analysis suggests that TP53 alteration is a valuable predictor for the prognosis of patients with H3K27M mutated gliomas. TP53 alteration may be used for identifying a subset of patients who potentially benefit from targeted reactivation of TP53 activity.