A knockout-first model of H3f3a gene targeting leads to developmental lethality.

Histone variant H3.3 is encoded by two genes, H3f3a and H3f3b, which can be expressed differentially depending on tissue type. Previous work in our lab has shown that knockout of H3f3b causes some neonatal lethality and infertility in mice, and chromosomal defects in mouse embryonic fibroblasts (MEFs). Studies of H3f3a and H3f3b null mice by others have produced generally similar phenotypes to what we found in our H3f3b nulls, but the relative impacts of the loss of either H3f3a or H3f3b have varied depending on the approach and genetic background. Here we used a knockout-first approach to target the H3f3a gene for inactivation in C57BL6 mice. Homozygous H3f3a targeting produced a lethal phenotype at or before birth. E13.5 null embryos had some potential morphological differences from WT littermates including smaller size and reduced head size. An E18.5 null embryo was smaller than its control littermates with several potential defects including small head and brain size as well as small lungs, which would be consistent with a late gestation lethal phenotype. Despite a reduction in H3.3 and total H3 protein levels, the only histone H3 post-translational modification in the small panel assessed that was significantly altered was the unique H3.3 mark phospho-Serine31, which was consistently increased in null neurospheres. H3f3a null neurospheres also exhibited consistent gene expression changes including in protocadherins. Overall, our findings are consistent with the model that there are differential, cell-type-specific contributions of H3f3a and H3f3b to H3.3 functions in epigenetic and developmental processes.

A comprehensive genomic study of 390 H3F3A-mutant pediatric and adult diffuse high-grade gliomas, CNS WHO grade 4.

Malignant brain tumors, known as H3K27-altered diffuse midline glioma (DMG) and H3G34-mutant diffuse hemispheric glioma (DHG), can affect individuals of all ages and are classified as CNS WHO grade 4. We comprehensively characterized 390 H3F3A-mutant diffuse gliomas (201 females, 189 males) arising in pediatric patients (under 20 years old) and adults (20 years and older) evaluated by the CGP program at Foundation Medicine between 2013 and 2020. We assessed information from pathology reports, histopathology review, and clinical data. The cohort included 304 H3K27M-mutant DMG (156 females, 148 males) and 86 H3G34-mutant DHG (45 females, 41 males). Median patient age was 20 years (1-74 years). The frequency of H3K27M-mutant DMG was similar in both pediatric and adult patients in our cohort-48.6% of the patients were over 20 years old, 31.5% over 30, and 18% over 40 at initial diagnosis. FGFR1 hotspot point mutations (N546K and K656E) were exclusively identified in H3K27M-mutant DMG tumors (64/304, 21%; p = 0.0001); these tend to occur in older patients (median age: 32.5 years) and mainly arose in the diencephalon. H3K27M-mutant DMG had higher rates of mutations in NF1 (31.0 vs 8.1%; p = 0.0001) and PIK3CA/PIK3R1 (27.9% vs 15.1%; p = 0.016) compared to H3G34-mutant DHG. However, H3G34-mutant DHG had higher rates of targetable alterations in cell-cycle pathway genes (CDK4 and CDK6 amplification; CDKN2A/B deletion) (27.0 vs 9.0%). Potentially targetable PDGFRA alterations were identified in ~ 20% of both H3G34-mutant DHG and H3K27M-mutant DMG. Overall, in the present study H3K27M-mutant DMG occurred at similar rates in both adult and patient patients. Through our analysis, we were able to identify molecular features characteristic of DMG and DHG. By identifying the recurrent co-mutations including actionable FGFR1 point mutations found in nearly one-third of H3K27M-mutant DMG in young adults, our findings can inform clinical translational studies, patient diagnosis, and clinical trial design.

Epigenetic lockdown of CDKN1A (p21) and CDKN2A (p16) characterises the neoplastic spindle cell component of giant cell tumours of bone.

Giant cell tumour of bone (GCTB) comprises the eponymous osteoclastic multinucleated giant cells eliciting bone lysis, an H3F3A-mutated neoplastic mononucleated fibroblast-like cell population, and H3F3A wild-type mononucleated stromal cells. In this study, we characterised four new cell lines from GCTB. Furthermore, we compared the genome-wide DNA methylation profile of 13 such tumours and three further cell lines with giant cell-rich lesions comprising three H3F3B-mutated chondroblastomas, three USP6-rearranged aneurysmal bone cysts, three non-ossifying fibromas, two hyperparathyroidism-associated brown tumours as well as mesenchymal stem cells, osteoblasts, and osteoclasts. In an unsupervised analysis, we delineated GCTB and chondroblastomas from the other analysed tumour entities. Using comparative methylation analysis, we demonstrated that the methylation pattern of the cell lines approximately equals that of H3F3A-mutated stromal cells in tissue. These patterns more resemble that of osteoblasts than that of mesenchymal stem cells, which argues for the osteoblast as the cell of origin of giant cell tumours of bone. Using enrichment analysis, we detected distinct hypermethylated clusters containing histone and collagen genes as well as target genes of the tumour suppressor p53. We found that the promotor regions of CDKN1A, CDKN2A, and IGFBP3 are methylated more strongly in GCTB than in the other giant cell-containing lesions, mesenchymal stem cells, osteoblasts, and osteoclasts (p < 0.001). This hypermethylation correlates with the lower gene expression at the mRNA level for these three genes in the cell lines, the lack of p16 and p21 in these cell lines, and the lower expression of p16 and p21 in GCTB. Overall, our analysis reveals characteristic DNA methylation patterns of giant cell tumours of bone and chondroblastomas and shows that cell lines of giant cell tumours of bone are a valid model for further analysis of H3F3A-mutated tumour cells. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Giant-cell-poor giant cell tumor of bone: report of two cases and literature review.

Giant cell tumor of bone (GCTB) is a locally aggressive tumor that shows predilection for the metaphysis/epiphysis of long bones, with an incidence of 4-5% of primary bone tumors. GCTB shows two main populations of cells: mononuclear cells and non-neoplastic multi-nucleated giant cells, with or without fibrous background. On the other hand, giant-cell-poor GCTB are rare with only few reports in the literature. These cases offer a diagnostic challenge, given the absence of giant cells and such cases have consistently been shown to harbor the H3F3A gene mutation by sequencing. The H3.3 G34W mutation-specific monoclonal antibody has shown high specificity in the diagnosis of GCTB. Two cases of giant-cell-poor GCTB are presented in this study, in which giant cells were absent or sparse and the diagnosis of GCTB was confirmed by the expression of H3.3 G34W monoclonal antibody in the mononuclear cells by immunohistochemistry. Whether this represents a histologic variant of GCTB or partial involution of GCTB is not yet fully understood; however, an immune response, infectious/inflammatory reaction, and/or anti-tumor cytokine production have been purported to be factors inciting disease regression in GCTB.

Bone and soft tissue tumors: clinicoradiologic-pathologic molecular-genetic correlation of novel fusion spindled, targetable-ovoid, giant-cell-rich, and round cell sarcomas.

BACKGROUND: New entities in the classification of bone and soft tissue tumors have been identified by use of advanced molecular-genetic techniques, including next-generation sequencing. Clinicoradiologic and pathologic correlation supports diagnostic classification. METHODS: Tumors from four morphologically grouped areas are selected to enhance diagnosis and awareness among the multidisciplinary team. These include select round cell tumors, spindle cell tumors, targetable tyrosine kinase/RAS::MAPK pathway-ovoid (epithelioid to spindled) tumors, and giant-cell-rich tumors of bone and soft tissue. RESULTS: Round cell tumors of bone and soft tissue include prototypical Ewing sarcoma, newer sarcomas with BCOR genetic alteration and CIC-rearranged, as well as updates on FUS/EWSR1::NFATc2, an EWSR1 non-ETS tumor that is solid with additional amplified hybridization signal pattern of EWSR1. This FUS/EWSR1::NFATc2 fusion has now been observed in seemingly benign to low-grade intraosseous vascular-rich and simple (unicameral) bone cyst tumors. Select spindle cell tumors of bone and soft tissue include rhabdomyosarcoma with FUS/EWSR1::TFCP2, an intraosseous high-grade spindle cell tumor without matrix. Targetable tyrosine-kinase or RAS::MAPK pathway-tumors of bone and soft tissue include NTRK, ALK, BRAF, RAF1, RET, FGFR1, ABL1, EGFR, PDGFB, and MET with variable ovoid myopericytic to spindled pleomorphic features and reproducible clinicopathologic and radiologic clues to their diagnosis. Giant-cell-rich tumors of bone, joint, and soft tissue are now respectively characterized by H3F3A mutation, CSF1 rearrangement (targetable), and HMGA2::NCOR2 fusion. CONCLUSION: This article is an update for radiologists, oncologists, surgeons, and pathologists to recognize these novel ovoid, spindled, giant-cell-rich, and round cell tumors, for optimal diagnostic classification and multidisciplinary team patient care.

The prognostic significance of HIST1H3B/C and H3F3A K27M mutations in diffuse midline gliomas is influenced by patient age.

INTRODUCTION: Diffuse midline gliomas (DMGs) are infiltrative midline gliomas harboring H3K27M mutations and are generally associated with poor outcomes. H3K27M mutations include mutations in HIST1H3B/C (H3.1), HIST2H3B/D (H3.2), or H3F3A (H3.3) genes. It is still unclear whether these mutations each portend a universally poor prognosis, or if there are any factors which modulate outcome. The main objective of this study was to study overall survival (OS) of H3.1 versus H3.3 K27M-mutant DMGs in pediatric and adult patients. METHODS: PubMed and Web of Science were searched, and we included studies if they have individual patient data of DMGs with available H3K27M genotype. Kaplan-Meier analysis and Cox regression models were used to analyze the survival of H3.1 and H3.3 mutations in each subgroup. RESULTS: We included 26 studies with 102 and 529 H3.1 and H3.3-mutant DMGs, respectively. The H3.1 mutation was more commonly seen in younger age. In pediatric population, H3.3 mutation conferred a shorter survival (median OS of 10.1 vs 14.2 months; p < 0.001) in comparison to H3.1-positive patients, which was further confirmed in the multivariate Cox analysis. Conversely, H3.3 was associated with a prolonged survival in adult patients as compared with H3.1 mutation (median OS of 14.4 vs 1.7 months; p = 0.019). CONCLUSION: We demonstrated that the prognosis of H3.1 and H3.3 K27M mutation in DMG patients is modulated by patient age. Routine H3K27M mutation genotyping in newly diagnosed DMGs may further stratify patients with these difficult tumors.

Clinicopathologic and molecular features of denosumab-treated giant cell tumour of bone (GCTB): Analysis of 21 cases.

GCTB is an osteolytic, locally-aggressive, rarely-metastasizing tumour, characterized by abundance of osteoclast-like giant cells, induced by neoplastic mononuclear cells expressing high-levels of the receptor activator of nuclear factor Kappa-B ligand (RANKL), a mediator of osteoclast activation. Although the mainstay of treatment is complete tumour removal with preservation of bone, therapy with denosumab, an inhibitor of RANKL, has been introduced for selected cases. OBJECTIVES: Denosumab-treated GCTB (DT-GCTB) was reported to show a wide spectrum of histological changes such as depletion of osteoclast-like giant cells and intralesional bone deposition, which may lead to diagnostic difficulties. We investigated clinicopathologic and molecular features of DT-GCTB, matched with pre-therapy samples. PARTICIPANTS: 21 cases were included (13 females, 8 males), aged 15 to 64 (median, 30 years). RESULTS: DT-GCTB showed development of sclerotic neocortex and varying degrees of osteosclerosis radiographically. Marked depletion of giant cells, different degree of ossification, fibrosis, and proliferation of mononuclear cells was observed. Staining for H3.3G34W was positive in mononuclear cells in 19 cases (90.5%), while one negative case was positive for H3.3G34V. H3F3A G34W mutation was confirmed in 17 of 19 cases (89.5%), corresponding to nuclear staining with H3.3 G34W antibody. G34L mutation was detected in one G34W negative case, in which H3.3 G34V nuclear positive staining was observed, possibly due to cross-reaction. CONCLUSIONS: Post-therapy tumours still exhibit a similar mutation profile, while significantly differing from classic GCTB morphologically. Correlation with history of denosumab administration, awareness of features of DT-GCTB, IHC and molecular studies for histone H3 mutations are important in its assessment.

Malignant Transformation of Giant Cell Tumour of Bone: A Review of Literature and the Experience of a Referral Centre.

Giant cell tumour of bone (GCTB) is a benign, locally aggressive primary bone neoplasm that represents 5% of all bone tumours. The principal treatment approach is surgery. Although generally GCTB is considered only a locally aggressive disease, it can metastasise, and lung metastases occur in 1-9% of patients. To date, only the use of denosumab has been approved as medical treatment for GCTB. Even more rarely, GCTB undergoes sarcomatous transformation into a malignant tumour (4% of all GCTB), but history of this malignant transformation is unclear and unpredictable. Considering the rarity of the event, the data in the literature are few. In this review, we summarise published data of GCTB malignant transformation and we analyse three cases of malignant transformation of GCTB, evaluating histopathology, genetics, and radiological aspects. Despite the rarity of this event, we conclude that a strict follow up is recommended to detect early malignant transformation.

Diagnostic Utility of Genetic and Immunohistochemical H3-3A Mutation Analysis in Giant Cell Tumour of Bone.

To validate the reliability and implementation of an objective diagnostic method for giant cell tumour of bone (GCTB). H3-3A gene mutation testing was performed using two different methods, Sanger sequencing and immunohistochemical (IHC) assays. A total of 214 patients, including 120 with GCTB and 94 with other giant cell-rich bone lesions, participated in the study. Sanger sequencing and IHC with anti-histone H3.3 G34W and G34V antibodies were performed on formalin-fixed, paraffin-embedded tissues, which were previously decalcified in EDTA if needed. The sensitivity and specificity of the molecular method was 100% (95% CI: 96.97-100%) and 100% (95% CI: 96.15-100%), respectively. The sensitivity and specificity of IHC was 94.32% (95% CI: 87.24-98.13%) and 100% (95% CI: 93.94-100.0%), respectively. P.G35 mutations were discovered in 2/9 (22.2%) secondary malignant GCTBs and 9/13 (69.2%) GCTB after denosumab treatment. We confirmed in a large series of patients that evaluation of H3-3A mutational status using direct sequencing is a reliable tool for diagnosing GCTB, and it should be incorporated into the diagnostic algorithm. Additionally, we discovered IHC can be used as a screening tool. Proper tissue processing and decalcification are necessary. The presence of the H3-3A mutation did not exclude malignant GCTB. Denosumab did not eradicate the neoplastic cell population of GCTB.

H3F3A-mutated giant cell tumour of bone without giant cells-clinical presentation, radiology and histology of three cases.

AIMS: Giant cell tumour of bone (GCTB) is histologically defined as a lesion containing reactive giant cells and a neoplastic mononuclear cell population; in up to 92% of cases, GCTB is characterised by a specific mutation of the histone gene H3F3A. The cellular composition ranges from giant-cell-rich to giant-cell-poor. The diagnosis of GCTB can be challenging, and several other lesions need to be excluded, e.g. aneurysmal bone cysts, non-ossifying fibromas, chondroblastomas, brown tumours, and giant-cell-rich osteosarcomas. Our aim was to analyse the clinical history, imaging, molecular pathology and histology of three H3F3A-mutated bone tumours without detectable giant cells. None of the patients received denosumab therapy. METHODS AND RESULTS: Diagnostic material was obtained by curettage or resection and/or biopsy. Common histomorphological features of all three reported lesions were fibrocytic, oval cells in a background of osteoid and an absence of multinuclear giant cells as confirmed with CD68 immunohistochemistry. We used immunohistochemistry and Sanger sequencing to demonstrate positivity for the H3.3 p.G34W mutation. Differential diagnoses were systematically excluded on the basis of histomorphology, immunohistochemistry, and fluorescence in-situ hybridisation. The imaging (radiography, computed tomography, and magnetic resonance imaging) for all three cases is presented and discussed. CONCLUSIONS: We believe that these GCTBs without giant cells expand one end of the heterogeneous range of GCTB. Because of the lack of giant cells, correct diagnosis of GCTB is challenging or even impossible on histological grounds alone. In these cases, detection of the characteristic H3F3A mutation (G34W-specific antibody RM263 or sequencing) is extremely helpful for diagnosing those lesions without giant cells as giant cell tumours of bone.

H3K27M-mutant diffuse midline gliomas should be further molecularly stratified: an integrated analysis of 669 patients.

INTRODUCTION: H3K27M-mutated diffuse midline gliomas (H3-DMGs) are aggressive tumors with a fatal outcome. This study integrating individual patient data (IPD) from published studies aimed to investigate the prognostic impact of different genetic alterations on survival of these patients. METHODS: We accessed PubMed and Web of Science to search for relevant articles. Studies were included if they have available data of follow-up and additional molecular investigation of H3-DMGs. For survival analysis, Kaplan-Meier analysis and Cox regression models were utilized, and corresponding hazard ratios (HR) and 95% confidence intervals (CI) were computed to analyze the impact of genetic events on overall survival (OS). RESULT: We included 30 studies with 669 H3-DMGs. TP53 mutations were the most common second alteration among these neoplasms. In univariate Cox regression model, TP53 mutation was an indicator of shortened survival (HR 1.446; 95% CI 1.143-1.829) whereas ACVR1 (HR 0.712; 95% CI 0.518-0.976) and FGFR1 mutations (HR 0.408; 95% CI 0.208-0.799) conferred prolonged survival. In addition, ATRX loss was also associated with a better OS (HR 0.620; 95% CI 0.386-0.996). Adjusted for age, gender, and tumor location, the presence of TP53 mutations, the absence of ACVR1 or FGFR1 mutations remained significantly poor prognostic factors. CONCLUSIONS: We outlined the prognostic importance of additional genetic alterations in H3-DMGs and recommended that these neoplasms should be further molecularly segregated. This may aid neuro-oncologists in appropriate risk stratification.

Bilateral Lung Metastases From a Phalangeal Giant Cell Tumor of Bone.

We describe a rare pediatric case of a phalangeal giant cell tumor of bone with extensive bilateral lung metastases following curettage, wide resection, and amputation. Concurrent peripheral blood eosinophilia and pleural effusion with marked eosinophilia (47%) were present. To discover genetic changes driving tumor metastasis, genomic and transcriptome profiling of the metastatic lung mass as well as germline analysis were performed. Whole exome sequencing detected a histone H3F3A p.G35V missense mutation in tumor cells. RNA sequencing revealed overexpression of receptor activator of nuclear factor kappa-B ligand (RANKL). The patient is alive with no residual disease and uncompromised respiratory function 29 months after amputation of primary tumor and 19 months after surgical resection of his metastatic lung disease.

The role of neuropathology in the management of newly diagnosed glioblastoma: a systematic review and evidence-based clinical practice guideline.

TARGET POPULATION: These recommendations apply to adult patients with newly diagnosed or suspected glioblastoma (GBM) QUESTION : For adult patients with newly diagnosed GBM does testing for Isocitrate Dehydrogenase 1 or 2 (IDH 1/2) mutations afford benefit beyond standard histopathology in providing accurate classification and outcome prognostication? Level III IDH 1/2 mutational status by immunohistochemistry (IHC) and/or sequencing is suggested for classification and prognostic information. Level III Non-canonical IDH 1/2 mutations are very rare in patients aged 55 or older and universal testing of variant mutations by sequence analysis is not suggested for this age range. QUESTION: For adult patients with lower grade infiltrating astrocytomas (WHO grades II and III) can the IDH-wildtype status designation supersede histopathology to predict prognosis and biologic relevance to eventual behavior as a GBM? Level III The designation of infiltrating astrocytomas (WHO grades II and III) as IDH-wildtype is not suggested as sufficient for a higher grade designation alone. Level III It is suggested that IDH-wildtype WHO grades II and III astrocytomas be tested for molecular-genetic alterations typical of IDH-wildtype GBM such as EGFR amplification, gain of chromosome 7/loss of chromosome 10 and TERT-p mutation to substantiate prediction of behavior similar to IDH-wildtype glioblastoma. Level III It is suggested that a diagnosis of diffuse astrocytic glioma, IDH-wildtype, with molecular features of GBM, WHO grade IV be rendered for infiltrating astrocytomas that lack histologic criteria of GBM but harbors molecular-genetic alterations of IDH-wildtype glioblastoma. QUESTION: For adult patients with newly diagnosed infiltrating glioma arising in the midline does testing for H3-K27M mutations provide information beyond that gained by histopathology for accurate classification and outcome prognostication? Level III It is suggested that infiltrating gliomas arising in midline anatomic locations be tested for the H3-K27M mutation as they tend to exhibit WHO grade IV behavior even if they lack histologic criteria for glioblastoma.

Retrospective analysis on the consistency of MRI features with histological and molecular markers in diffuse intrinsic pontine glioma (DIPG).

PURPOSE: The diagnosis of diffuse intrinsic pontine glioma (DIPG) is based largely on a combination of clinical and radiological findings due to the difficulty of obtaining a biopsy. An accurate evaluation of magnetic resonance imaging (MRI) scans is consequently essential. Recent analyses on the genomic landscape of DIPG revealed recurrent mutations in the H3F3A and HIST1H3B histone genes. We reviewed cases with available tumor tissue from institutional DIPG series to ascertain the consistency between their histo-molecular findings and clinical-radiological features. METHODS: We conducted a radiological and pathological central review of 22 cases enrolled in institutional DIPG trials. We performed immunohistochemical analyses to detect H3F3A/HIST1H3B K27M mutations, histone trimethylation, and EZH2 expression. Mutational analysis was performed for ACVR1, H3F3A, and HIST1H3B genes. RESULTS: Patients' median age at diagnosis was 8 years, and their median overall survival was 11 months. Nineteen/22 cases (86%) showed evidence of K27M mutation on immunohistochemistry and/or mutation analysis. Histone trimethylation expression was low or lacking in these mutated cases. Sequence analysis revealed 13 cases with H3F3A and 1 case with HIST1H3B K27M mutation. There was no significant difference in EZH2 expression between the K27M mutant and wild-type DIPGs. Upon external, blinded MRI re-evaluation one lesion not consistent with DIPG showed no evidence of K27M mutation and retained histone trimethylation expression. CONCLUSION: In conclusion, our study demonstrates a high frequency of histone K27M mutations in DIPG when MRI features are carefully assessed, thus confirming the consistency of imaging with biological markers in our institutional series of DIPG.

Histone H3.3 mutation in giant cell tumor of bone: an update in pathology.

Giant cell tumor of bone (GCTB) is a locally aggressive bone tumor that frequently shows local recurrence and occasionally shows malignant transformation to high-grade sarcoma. Histologically, conventional GCTB is composed mainly of three types of cells: mononuclear neoplastic cells with an osteoblastic precursor phenotype, mononuclear histiocytic cells, and osteoclast-like multinucleated giant cells. These cells interact with each other via the RANKL-RANK axis and other mechanisms for tumor formation. The vast majority of GCTBs were recently revealed to harbor H3F3A p.G34W mutation, and a minor subset have H3F3A p.G34L, p.G34M, p.G34R, or p.G34V mutation. H3.3 G34W mutant-specific immunohistochemistry is a highly sensitive and specific surrogate marker for H3F3A p.G34W mutation in GCTB and thus useful for differential diagnoses of histological mimics. H3.3 mutant-specific immunohistochemistry has also contributed to the understanding of the bone-forming ability of neoplastic cells of GCTB and the remarkable new bone formation after treatment with denosumab, an inhibitor of RANKL. In primary and secondary malignant GCTBs, the H3F3A gene allele can be preserved or lost with malignant transformation.

Telomere elongation via alternative lengthening of telomeres (ALT) and telomerase activation in primary metastatic medulloblastoma of childhood.

PURPOSE: Elongation of telomeres is necessary for tumor cell immortalization and senescence escape; neoplastic cells use to alternative pathways to elongate telomeres: telomerase reactivation or a telomerase-independent mechanism termed alternative lengthening of telomeres (ALT). Telomerase and ALT pathway has been explored in adult and pediatric gliomas and medulloblastomas (MDBs); however, these mechanisms were not previously investigated in MDBs metastatic at the onset. Therefore, we analyzed the activation of telomerase and ALT pathway in a homogenous cohort of 43 pediatric metastatic medulloblastomas, to investigate whether telomere elongation could play a role in the biology of metastatic MDB. METHODS: We evaluated telomeres length via telomere-specific fluorescence in situ hybridization (Telo-FISH); we assessed nuclear expression of ATRX by immunohistochemistry (IHC). H3F3A and TERT promoter mutations were analyzed by pyrosequencing, while UTSS methylation status was analyzed via methylation-specific-PCR (MS-PCR). RESULTS: H3F3A mutations were absent in all MDBs, 30% of samples showed ATRX nuclear loss, 18.2% of cases were characterized by TERT promoter mutations, while 60.9% harboured TERT promoter hyper-methylation in the UTSS region. Elongation of telomeres was found in 42.8% of cases. Metastatic MDBs control telomere elongation via telomerase activation (10.7%), induced by TERT promoter mutations in association with UTSS hyper-methylation, and ALT mechanism (32.1%), triggered by ATRX inactivation. Among non-metastatic MDBs, only 5.9% (1/17) showed ATRX nuclear loss with activation of ALT. CONCLUSIONS: Our metastatic cases frequently activate ALT pathway, suggesting that it is a common process for senescence escape in primary metastatic medulloblastomas. Furthermore, the activation of mechanisms for telomere elongation is not restricted to certain molecular subgroups in this high-risk group of MDBs.

Prognostic significance of genetic biomarkers in isocitrate dehydrogenase-wild-type lower-grade glioma: the need to further stratify this tumor entity - a meta-analysis.

The clinical outcomes of isocitrate dehydrogenase-wild-type (IDH-wt) lower-grade glioma (LGG) have been the subject of debate for some time. In this meta-analysis, we aimed to assess the prognostic values of several known genetic markers (e.g. TERT promoter mutation, H3F3A mutation, CDKN2A loss) in this tumor group. Four electronic databases, including PubMed, Scopus, Web of Science and Virtual Health Library, were searched for relevant articles. Pooled hazard ratio (HR) and corresponding 95% confidence interval (CI) for overall survival were calculated using a random-effect model weighted by an inverse variance method. A total of 11 studies were finally selected from 2274 articles for meta-analyses. Several genetic alterations were demonstrated to have a negative impact on prognosis of IDH-wt LGGs, specifically TERT promoter mutation (HR, 1.96; 95% CI, 1.42-2.70), H3F3A mutation (HR, 3.21; 95% CI, 1.86-5.55) and EGFR amplification (HR, 1.67; 95% CI, 1.02-2.74). However, CDKN loss, ATRX mutation and coexisting gain of chromosome 7/loss of chromosome 10 showed no clinical significance in this glioma entity. Our study results demonstrated that IDH-wt LGGs are heterogeneous in clinical outcome and not all tumors have a poor prognosis. The presence of TERT promoter mutation, H3F3A mutation and EGFR amplification showed negative prognostic impacts in this tumor entity. These genetic events can be used to better stratify patient outcomes.

Clinical Next Generation Sequencing Reveals an H3F3A Gene as a New Potential Gene Candidate for Microcephaly Associated with Severe Developmental Delay, Intellectual Disability and Growth Retardation.

Microcephaly is characterized by significant clinical and genetic heterogeneity, therefore reaching the genetic diagnosis remains challenging in this group of disorders. We describe a case of a girl with secondary microcephaly, associated with severe developmental delay, intellectual disability, growth retardation and dysmorphic features. For purposes of clinical genetic diagnostic testing, we performed trio whole exome sequencing in the proband and unaffected parents. We found a heterozygous de novo missense variant in the H3F3A gene in the proband (NM_ 002107.4: c.185T>G), which is absent from the gnomAD and from the Slovenian Genome databases. The identified variant affects a highly conserved leucine residue at position 62 of the histone H3 protein (H3.3) and is predicted to affect the physicochemical properties of the affected protein. Mouse models, which demonstrated involvement of H3.3 protein in the control of neuronal- and glial-specific gene expression patterns that control synaptic connectivity and behavioral plasticity. Additionally, we also identified similar cases reported in the ClinVar database. These arguments support the possible pathogenic role of the reported genetic variant and thus suggest a novel molecular mechanism for this syndromic form of microcephaly.

Chondroblastoma-like primary malignant giant cell tumor of the humerus - a case report.

35-year-old woman suffered prolonged pain in the left shoulder, where an aggressively growing tumor of the proximal humerus was revealed thereafter. The lesion caused massive osteolysis of the metaepiphysis with cortical disruption, but no soft tissue extension was evident. Given the unsatisfactory effect, the ongoing neoadjuvant chemotherapy was prematurely ceased and the resection 13 cm long segment of bone with modular prosthesis replacement followed. Histologically, clear-cut malignant tumor with both the presence of numerous reactive osteoclast-like giant cells and geographic structural deposition of chondroid matrix bore a close resemblance to chondroblastoma. Dominant cellular composition formed solid mosaic clusters of large, atypical, frequently binucleated cells with voluminous eosinophilic cytoplasm. Impressive nuclear pleomorphism was accentuated by both the grooving and atypical mitotic figures. Thorough sampling disclosed limited, but sharply contrasting parts, where biphasic arrangement of small uniform stromal elements together with regularly distributed, reactive osteoclasts suggested putative precursor giant cell lesion. Except the osteoclasts, all matrical and stromal cells were strongly SOX9 and D2-40 positive; in contrary desmin, SATB2, S100 and p63 yielded completely negative results. Detected H3F3A c.103G>T mutation in exon 2 finally established true nature of that peculiar neoplastic proliferation and lead to descriptive term of primary chondroblastoma-like malignant giant cell tumor. In the setting of all the microscopic variability, histogenesis and complex differential diagnosis of skeletal (malignant) giant cell lesions, there are discussed e.g. aggressive/malignant chondroblastoma, chondroblastoma-like osteosarcoma or giant cell-rich osteosarcoma and practical impact of specific mutational analysis results as well.

Pediatric low-grade gliomas can be molecularly stratified for risk.

Pediatric low-grade gliomas (PLGGs) consist of a number of entities with overlapping histological features. PLGGs have much better prognosis than the adult counterparts, but a significant proportion of PLGGs suffers from tumor progression and recurrence. It has been shown that pediatric and adult low-grade gliomas are molecularly distinct. Yet the clinical significance of some of newer biomarkers discovered by genomic studies has not been fully investigated. In this study, we evaluated in a large cohort of 289 PLGGs a list of biomarkers and examined their clinical relevance. TERT promoter (TERTp), H3F3A and BRAF V600E mutations were detected by direct sequencing. ATRX nuclear loss was examined by immunohistochemistry. CDKN2A deletion, KIAA1549-BRAF fusion, and MYB amplification were determined by fluorescence in situ hybridization (FISH). TERTp, H3F3A, and BRAF V600E mutations were identified in 2.5, 6.4, and 7.4% of PLGGs, respectively. ATRX loss was found in 4.9% of PLGGs. CDKN2A deletion, KIAA1549-BRAF fusion and MYB amplification were detected in 8.8, 32.0 and 10.6% of PLGGs, respectively. Survival analysis revealed that TERTp mutation, H3F3A mutation, and ATRX loss were significantly associated with poor PFS (p < 0.0001, p < 0.0001, and p = 0.0002) and OS (p < 0.0001, p < 0.0001, and p < 0.0001). BRAF V600E was associated with shorter PFS (p = 0.011) and OS (p = 0.032) in a subset of PLGGs. KIAA1549-BRAF fusion was a good prognostic marker for longer PFS (p = 0.0017) and OS (p = 0.0029). MYB amplification was also a favorable marker for a longer PFS (p = 0.040). Importantly, we showed that these molecular biomarkers can be used to stratify PLGGs into low- (KIAA1549-BRAF fusion or MYB amplification), intermediate-I (BRAF V600E and/or CDKN2A deletion), intermediate-II (no biomarker), and high-risk (TERTp or H3F3A mutation or ATRX loss) groups with distinct PFS (p < 0.0001) and OS (p < 0.0001). This scheme should aid in clinical decision-making.