Pediatric glioblastomas (GBM) including diffuse intrinsic pontine gliomas (DIPG) are devastating brain tumors with no effective therapy. Here, we investigated clinical and biological impacts of ...histone H3.3 mutations. Forty-two DIPGs were tested for H3.3 mutations. Wild-type versus mutated (K27M-H3.3) subgroups were compared for
HIST1H3B
,
IDH
,
ATRX
and
TP53
mutations, copy number alterations and clinical outcome. K27M-H3.3 occurred in 71 %,
TP53
mutations in 77 % and
ATRX
mutations in 9 % of DIPGs.
ATRX
mutations were more frequent in older children (
p
< 0.0001). No G34V/R-H3.3, IDH1/2 or H3.1 mutations were identified. K27M-H3.3 DIPGs showed specific copy number changes, including all gains/amplifications of
PDGFRA
and
MYC/PVT1
loci. Notably, all long-term survivors were H3.3 wild type and this group of patients had better overall survival. K27M-H3.3 mutation defines clinically and biologically distinct subgroups and is prevalent in DIPG, which will impact future therapeutic trial design. K27M- and G34V-H3.3 have location-based incidence (brainstem/cortex) and potentially play distinct roles in pediatric GBM pathogenesis. K27M-H3.3 is universally associated with short survival in DIPG, while patients wild-type for H3.3 show improved survival. Based on prognostic and therapeutic implications, our findings argue for H3.3-mutation testing at diagnosis, which should be rapidly integrated into the clinical decision-making algorithm, particularly in atypical DIPG.
Arthropods are vectors for many pathogens that significantly harm human and animal health globally, and research into vector-borne diseases is of critical public health importance. Arthropods present ...unique risks for containment, and therefore insectary facilities are essential to the safe handling of arthropod-borne hazards. In 2018, the School of Life Sciences at Arizona State University (ASU) began the process to build a level 3 arthropod containment (ACL-3) facility. Even with the COVID-19 pandemic, it took more than 4 years for the insectary to be granted a Certificate of Occupancy. At the request of the ASU Environmental Health and Safety team, Gryphon Scientific, an independent team with biosafety and biological research expertise, studied the project lifecycle through the design, construction, and commissioning of the ACL-3 facility with the goal of identifying lessons learned from the delayed timeline. These lessons learned convey insight into best practices for assessing potential facility sites, anticipating challenges with retrofitted construction, preparing for commissioning, equipping the project team with necessary expertise and expectations, and supplementing the gaps in available containment guidance. Several unique mitigations designed by the ASU team to address research risks not specifically addressed in the American Committee of Medical Entomology Arthropod Containment Guidelines are also described. Completion of the ACL-3 insectary at ASU was delayed, but the team thoroughly assessed potential risks and enabled appropriate practices for the safe handling of arthropod vectors. These efforts will enhance future ACL-3 construction by helping to avoid similar setbacks and streamlining progress from concept to operation.
Gliomas are the most common primary brain tumors in children and adults. We recently identified frequent alterations in chromatin remodelling pathways including recurrent mutations in
H3F3A
and ...mutations in
ATRX
(α-thalassemia/mental-retardation-syndrome-X-linked) in pediatric and young adult glioblastoma (GBM, WHO grade IV astrocytoma).
H3F3A
mutations were specific to pediatric high-grade gliomas and identified in only 3.4 % of adult GBM. Using sequencing and/or immunohistochemical analyses, we investigated ATRX alterations (mutation/loss of expression) and their association with
TP53
and
IDH1
or
IDH2
mutations in 140 adult WHO grade II, III and IV gliomas, 17 pediatric WHO grade II and III astrocytomas and 34 pilocytic astrocytomas. In adults, ATRX aberrations were detected in 33 % of grade II and 46 % of grade III gliomas, as well as in 80 % of secondary and 7 % of primary GBMs. They were absent in the 17 grade II and III astrocytomas in children, and the 34 pilocytic astrocytomas. ATRX alterations closely overlapped with mutations in
IDH1
/
2
(
p
< 0.0001) and
TP53
(
p
< 0.0001) in samples across all WHO grades. They were prevalent in astrocytomas and oligoastrocytomas, but were absent in oligodendrogliomas (
p
< 0.0001). No significant association of ATRX mutation/loss of expression and alternative lengthening of telomeres was identified in our cohort. In summary, our data show that ATRX alterations are frequent in adult diffuse gliomas and are specific to astrocytic tumors carrying
IDH1
/
2
and
TP53
mutations. Combined alteration of these genes may contribute to drive the neoplastic growth in a major subset of diffuse astrocytomas in adults.
Measurement of liver iron concentration (LIC) is necessary for a range of iron-loading disorders such as hereditary hemochromatosis, thalassemia, sickle cell disease, aplastic anemia, and ...myelodysplasia. Currently, chemical analysis of needle biopsy specimens is the most common accepted method of measurement. This study presents a readily available noninvasive method of measuring and imaging LICs in vivo using clinical 1.5-T magnetic resonance imaging units. Mean liver proton transverse relaxation rates (R2) were measured for 105 humans. A value for the LIC for each subject was obtained by chemical assay of a needle biopsy specimen. High degrees of sensitivity and specificity of R2 to biopsy LICs were found at the clinically significant LIC thresholds of 1.8, 3.2, 7.0, and 15.0 mg Fe/g dry tissue. A calibration curve relating liver R2 to LIC has been deduced from the data covering the range of LICs from 0.3 to 42.7 mg Fe/g dry tissue. Proton transverse relaxation rates in aqueous paramagnetic solutions were also measured on each magnetic resonance imaging unit to ensure instrument-independent results. Measurements of proton transverse relaxivity of aqueous MnCl2 phantoms on 13 different magnetic resonance imaging units using the method yielded a coefficient of variation of 2.1%.
Recurrent mutations affecting the histone H3.3 residues Lys27 or indirectly Lys36 are frequent drivers of pediatric high-grade gliomas (over 30 % of HGGs). To identify additional driver mutations in ...HGGs, we investigated a cohort of 60 pediatric HGGs using whole-exome sequencing (WES) and compared them to 543 exomes from non-cancer control samples. We identified mutations in
SETD2
, a H3K36 trimethyltransferase, in 15 % of pediatric HGGs, a result that was genome-wide significant (FDR = 0.029). Most
SETD2
alterations were truncating mutations. Sequencing the gene in this cohort and another validation cohort (123 gliomas from all ages and grades) showed
SETD2
mutations to be specific to high-grade tumors affecting 15 % of pediatric HGGs (11/73) and 8 % of adult HGGs (5/65) while no
SETD2
mutations were identified in low-grade diffuse gliomas (0/45). Furthermore,
SETD2
mutations were mutually exclusive with
H3F3A
mutations in HGGs (
P
= 0.0492) while they partly overlapped with
IDH1
mutations (4/14), and
SETD2
-mutant tumors were found exclusively in the cerebral hemispheres (
P
= 0.0055). SETD2 is the only H3K36 trimethyltransferase in humans, and
SETD2
-mutant tumors showed a substantial decrease in H3K36me3 levels (
P
< 0.001), indicating that the mutations are loss-of-function. These data suggest that loss-of-function SETD2 mutations occur in older children and young adults and are specific to HGG of the cerebral cortex, similar to the H3.3 G34R/V and IDH mutations. Taken together, our results suggest that mutations disrupting the histone code at H3K36, including H3.3 G34R/V, IDH1 and/or SETD2 mutations, are central to the genesis of hemispheric HGGs in older children and young adults.
Migration of monocytes-macrophages plays an important role in phagocytosis of pathogens and cellular debris in a variety of pathophysiological conditions. Although epithelial Na
channels (ENaCs) are ...required for normal migratory responses in other cell types, their role in macrophage migration signaling is unknown. To address this possibility, we determined whether ENaC message is present in several peripheral blood monocyte cell populations and tissue-resident macrophages in healthy humans using the Human Protein Atlas database (www.proteinatlas.org) and the mouse monocyte cell line RAW 264.7 using RT-PCR. We then determined that selective ENaC inhibition with amiloride inhibited chemotactic migration (∼50%), but not phagocytosis, of the mouse monocyte-macrophage cell line RAW 264.7. Furthermore, we generated a cell line stably expressing an NH
-terminal truncated αENaC to interrupt normal channel trafficking and found it suppressed migration. Prolonged exposure (48 h) of RAW 264.7 cells to proinflammatory cytokines interferon γ (IFNγ) and/or tumor necrosis factor α (TNFα) inhibited RAW 264.7 migration and abolished the amiloride (1 µM)-sensitive component of migration, a finding consistent with ENaC downregulation. To determine if proinflammatory cytokines regulate αENaC protein expression, cells were exposed to proinflammatory cytokines IFNγ (10 ng/mL, last 48 h) and TNFα (10 ng/mL, last 24 h). By Western blot analysis, we found whole cell αENaC protein is reduced ≥50%. Immunofluorescence demonstrated heterogeneous αENaC inhibition. Finally, we found that overnight exposure to amiloride stimulated morphological changes and increased polarization marker expression. Our findings suggest that ENaC may be a critical molecule in macrophage migration and polarization.
Measurement of myocardial iron is key to the clinical management of patients at risk of siderotic cardiomyopathy. The cardiovascular magnetic resonance relaxation parameter R2* (assessed clinically ...via its reciprocal, T2*) measured in the ventricular septum is used to assess cardiac iron, but iron calibration and distribution data in humans are limited.
Twelve human hearts were studied from transfusion-dependent patients after either death (heart failure, n=7; stroke, n=1) or transplantation for end-stage heart failure (n=4). After cardiovascular magnetic resonance R2* measurement, tissue iron concentration was measured in multiple samples of each heart with inductively coupled plasma atomic emission spectroscopy. Iron distribution throughout the heart showed no systematic variation between segments, but epicardial iron concentration was higher than in the endocardium. The mean ± SD global myocardial iron causing severe heart failure in 10 patients was 5.98 ± 2.42 mg/g dry weight (range, 3.19 to 9.50 mg/g), but in 1 outlier case of heart failure was 25.9 mg/g dry weight. Myocardial lnR2* was strongly linearly correlated with lnFe (R²=0.910, P<0.001), leading to Fe=45.0×(T2*)⁻¹·²² for the clinical calibration equation with Fe in milligrams per gram dry weight and T2* in milliseconds. Midventricular septal iron concentration and R2* were both highly representative of mean global myocardial iron.
These data detail the iron distribution throughout the heart in iron overload and provide calibration in humans for cardiovascular magnetic resonance R2* against myocardial iron concentration. The iron values are of considerable interest in terms of the level of cardiac iron associated with iron-related death and indicate that the heart is more sensitive to iron loading than the liver. The results also validate the current clinical practice of monitoring cardiac iron in vivo by cardiovascular magnetic resonance of the midseptum.
Studies to date have yielded inconclusive results as to whether maternal medical history during pregnancy, and a child's early-life medical history contribute to the development of childhood brain ...tumours (CBTs). This study examined associations between maternal and childhood medical history and the risk of CBTs.
The Childhood Brain Tumour Epidemiology Study of Ontario (CBREO) examined children 0-15 years of age with newly diagnosed CBTs from 1997 to 2003. Multivariable logistic regression analysis determined associations for prenatal medications and childhood medical history, adjusted for child's demographics, and maternal education. Analyses were stratified by histology. A latency period analysis was conducted using 12- and 24-month lead times.
Maternal intake of immunosuppressants during the prenatal period was significantly associated with glial tumours (OR 2.73, 95% CI 1.17-6.39). Childhood intake of anti-epileptics was significantly associated with CBTs overall, after accounting for 12-month (OR 8.51, 95% CI 3.35-21.63) and 24-month (OR 6.04, 95% CI 2.06-17.70) lead time before diagnosis. No associations for other medications were found.
This study underscores the need to examine potential carcinogenic effects of the medication classes highlighted and of the indication of medication use. Despite possible reverse causality, increased CBT surveillance for children with epilepsy might be warranted.
The monocyte–macrophage system plays an important role in phagocytosis of pathogens and cellular debris following infection or tissue injury in several pathophysiological conditions. We examined ...ENaC/ASIC subunit transcript expression and the importance of select subunits in migration of bone marrow derived monocytes (freshly isolated) and macrophages (monocytes differentiated in culture). We also examined the effect of select subunit deletion on macrophage phenotype. BM monocytes were harvested from the femurs of male and female WT and KO mice (6–12 weeks of age). Our results show that α, β, γENaC, and ASIC1‐5 transcripts are expressed in BM macrophages and monocytes to varying degrees. At least αENaC, βENaC, and ASIC2 subunits contribute to chemotactic migration responses in BM monocyte–macrophages. Polarization markers (CD86, soluble TNFα) in BM macrophages from mice lacking ASIC2a plus βENaC were shifted towards the M1 phenotype. Furthermore, select M1 phenotypic markers were recovered with rescue of βENaC or ASIC2. Taken together, these data suggest that βENaC and ASIC2 play an important role in BM macrophage migration and loss of βENaC and/or ASIC2 partially polarizes macrophages to the M1 phenotype. Thus, targeting ENaC/ASIC expression in BM macrophages may regulate their ability to migrate to sites of injury.
Multidisciplinary neuro-oncology clinics allow collaboration between various specialties and training levels. Building a tenable clinical research program based in the longitudinal dialogue and ...practice of collaborative clinicians and trainees can bridge clinical observations to research execution. However, forming a research team around a multidisciplinary clinic's activities is constrained by a lack of literature or guidelines. As well, challenges in sustaining team logistics, communication, and productivity can persist without a standardized team framework. This perspective discusses the state of research teams in clinical oncology, and uses experiences from the McMaster Pediatric Brain Tumour Study Group to guide those seeking to form a research team based on the collective activities and observations of a multidisciplinary clinic.
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