With the number of prognostic and predictive genetic markers in neuro-oncology steadily growing, the need for comprehensive molecular analysis of neuropathology samples has vastly increased. We ...therefore developed a customized enrichment/hybrid-capture-based next-generation sequencing (NGS) gene panel comprising the entire coding and selected intronic and promoter regions of 130 genes recurrently altered in brain tumors, allowing for the detection of single nucleotide variations, fusions, and copy number aberrations. Optimization of probe design, library generation and sequencing conditions on 150 samples resulted in a 5-workday routine workflow from the formalin-fixed paraffin-embedded sample to neuropathological report. This protocol was applied to 79 retrospective cases with established molecular aberrations for validation and 71 prospective cases for discovery of potential therapeutic targets. Concordance of NGS compared to established, single biomarker methods was 98.0 %, with discrepancies resulting from one case where a
TERT
promoter mutation was not called by NGS and three ATRX mutations not being detected by Sanger sequencing. Importantly, in samples with low tumor cell content, NGS was able to identify mutant alleles that were not detectable by traditional methods. Information derived from NGS data identified potential targets for experimental therapy in 37/47 (79 %) glioblastomas, 9/10 (90 %) pilocytic astrocytomas, and 5/14 (36 %) medulloblastomas in the prospective target discovery cohort. In conclusion, we present the settings for high-throughput, adaptive next-generation sequencing in routine neuropathology diagnostics. Such an approach will likely become highly valuable in the near future for treatment decision making, as more therapeutic targets emerge and genetic information enters the classification of brain tumors.
Pediatric glioblastoma (pedGBM) is amongst the most common malignant brain tumors of childhood and carries a dismal prognosis. In contrast to adult GBM, few molecular prognostic markers for the ...pediatric counterpart have been established. We, therefore, investigated the prognostic significance of genomic and epigenetic alterations through molecular analysis of 202 pedGBM (1–18 years) with comprehensive clinical annotation. Routinely prepared formalin-fixed paraffin-embedded tumor samples were assessed for genome-wide DNA methylation profiles, with known candidate genes screened for alterations via direct sequencing or FISH. Unexpectedly, a subset of histologically diagnosed GBM (
n
= 40, 20 %) displayed methylation profiles similar to those of either low-grade gliomas or pleomorphic xanthoastrocytomas (PXA). These tumors showed a markedly better prognosis, with molecularly PXA-like tumors frequently harboring BRAF V600E mutations and 9p21 (
CDKN2A
) homozygous deletion. The remaining 162 tumors with pedGBM molecular signatures comprised four subgroups: H3.3 G34-mutant (15 %), H3.3/H3.1 K27-mutant (43 %), IDH1-mutant (6 %), and H3/IDH wild-type (wt) GBM (36 %). These subgroups were associated with specific cytogenetic aberrations,
MGMT
methylation patterns and clinical outcomes. Analysis of follow-up data identified a set of biomarkers feasible for use in risk stratification: pedGBM with any oncogene amplification and/or K27M mutation (
n
= 124) represents a particularly unfavorable group, with 3-year overall survival (OS) of 5 %, whereas tumors without these markers (
n
= 38) define a more favorable group (3-year OS ~70 %).Combined with the lower grade-like lesions, almost 40 % of pedGBM cases had distinct molecular features associated with a more favorable outcome. This refined prognostication method for pedGBM using a molecular risk algorithm may allow for improved therapeutic choices and better planning of clinical trial stratification for this otherwise devastating disease.
Hot spot mutations in the promoter region of telomerase reverse transcriptase (
TERT
) have recently been described in several human tumor entities. These mutations result in an upregulation of the ...telomerase complex activity and thus constitute a relevant mechanism for immortalization of tumor cells. Knowledge of the
TERT
promoter status in tumors is likely to be of interest for molecular classification and as a potential target for therapy. We, therefore, performed a systematic analysis of
TERT
promoter mutations in 1,515 tumors of the human nervous system and its coverings including 373 pediatric and 1,142 adult patients. We detected a total of 327 mutations.
TERT
promoter mutations were exceedingly rare in tumors typically encountered in pediatric patients. In entities typically encountered in adult patients
TERT
promoter mutations were strongly associated with older age (
p
< 0.0001). Highest mutation frequencies were detected in gliosarcomas (81 %), oligodendrogliomas (78 %), oligoastrocytomas (58 %), primary glioblastomas (54 %), and solitary fibrous tumors (50 %). Related to other molecular alterations,
TERT
promoter mutations were strongly associated with 1p/19q loss (
p
< 0.0001), but inversely associated with loss of ATRX expression (
p
< 0.0001) and
IDH1/IDH2
mutations (
p
< 0.0001).
TERT
promoter mutations are typically found in adult patients and occur in a highly tumor type-associated distribution.
In contrast to the relative morphological uniformity of histone H3 K27-mutant high-grade gliomas, H3 G34-mutant tumors present as a histopathologically heterogeneous group of neoplasms, with ...microscopic characteristics typical of either glioblastoma (GBM) or central nervous system primitive neuroectodermal tumors (CNS-PNET). In the current study, we performed an integrative clinical, histopathological and molecular analysis of 81 G34-mutant CNS tumors. Routinely prepared tumor tissues were investigated for genomic and epigenomic alterations. Despite their divergent histopathological appearance, CNS tumors with H3.3 G34 mutations displayed uniform epigenetic signatures, suggesting a single biological origin. Comparative cytogenetic analysis with other GBM subtypes disclosed a high frequency and high specificity of 3q and 4q loss across G34-mutant tumors.
PDGFRA
amplification was more common in cases with GBM than with PNET morphology (36 vs. 5 %, respectively), while
CCND2
amplifications showed the opposite trend (5 vs. 27 %). Survival analysis revealed the presence of amplified oncogene(s) and
MGMT
methylation as independent prognostic markers for poor and favorable outcomes, respectively. No difference in outcome was found between morphological variants (GBM vs. PNET). Thus, different histological variants of G34-mutant CNS tumors likely comprise a single biological entity (high-grade glioma with H3 G34 mutation, HGG_G34), which should be outlined in future diagnostic and therapeutic classifications. Screening for H3.3 G34 mutation should therefore be recommended as a routine diagnostic marker for supratentorial CNS tumors across a broad histological spectrum.
Animal manures, used as a nitrogen source for crop production, are often associated with negative impacts on nutrient levels in surface water. The concentrations of estrogens in streams from these ...manures also are of concern due to potential endocrine disruption in aquatic species. Streams associated with livestock operations were sampled by discrete samples (n = 38) or by time-integrated polar organic chemical integrative samplers (POCIS, n = 19). Samples were analyzed for estrogens by gas chromatography-tandem mass spectrometry (GC-MS2) and estrogenic activity was assessed by three bioassays: Yeast Estrogen Screen (YES), T47D-KBluc Assay, MCF-7 Estrogenicity Screen (E-Screen). Samples were collected from 19 streams within small (∼1–30 km2) watersheds in 12 U.S. states representing a range of hydrogeologic conditions, dominated by: dairy (3), grazing beef (3), feedlot cattle (1); swine (5); poultry (3); and 4 areas where no livestock were raised or manure was applied. Water samples were consistently below the United Kingdom proposed Lowest Observable Effect Concentration for 17β-estradiol in fish (10 ng/L) in all watersheds, regardless of land use. Estrogenic activity was often higher in samples during runoff conditions following a period of manure application. Estrone was the most commonly detected estrogen (13 of 38 water samples, mean 1.9, maximum 8.3 ng/L). Because of the T47D-KBluc assay's sensitivity towards estrone (1.4 times 17β-estradiol) it was the most sensitive method for detecting estrogens, followed by the E-Screen, GC-MS2, and YES. POCIS resulted in more frequent detections of estrogens than discrete water samples across all sites, even when applying the less-sensitive YES bioassay to the POCIS extracts.
Display omitted
•Estrogens and E2Equivalents in streams were below proposed LOEC for 17β-E2 in fish – no EE2 found.•E-Screen and T47D-KBluc bioassays more sensitive than GC-MS2 and YES.•T47D-KBluc was assay of choice for detection of most prevalent natural estrogen, estrone.•Passive samplers increased ability to detect the presence of estrogenic inputs.•Increased E2 may follow manure application or originate from non-livestock sources.
Activation of the MAPK signaling pathway has been shown to be a unifying molecular feature in pilocytic astrocytoma (PA). Genetically, tandem duplications at chromosome 7q34 resulting in
KIAA1549
–
...BRAF
fusion genes constitute the most common mechanism identified to date. To elucidate alternative mechanisms of aberrant MAPK activation in PA, we screened 125 primary tumors for
RAF
fusion genes and mutations in
KRAS
,
NRAS
,
HRAS
,
PTPN11
,
BRAF
and
RAF1
. Using microarray-based comparative genomic hybridization (aCGH), we identified in three cases an interstitial deletion of ~2.5 Mb as a novel recurrent mechanism forming
BRAF
gene fusions with
FAM131B
, a currently uncharacterized gene on chromosome 7q34. This deletion removes the BRAF N-terminal inhibitory domains, giving a constitutively active BRAF kinase. Functional characterization of the novel FAM131B–BRAF fusion demonstrated constitutive MEK phosphorylation potential and transforming activity in vitro. In addition, our study confirmed previously reported
BRAF
and
RAF1
fusion variants in 72% (90/125) of PA. Mutations in
BRAF
(8/125),
KRAS
(2/125) and
NF1
(4/125) and the rare
RAF1
gene fusions (2/125) were mutually exclusive with
BRAF
rearrangements, with the exception of two cases in our series that concomitantly harbored more than one hit in the MAPK pathway. In summary, our findings further underline the fundamental role of RAF kinase fusion products as a tumor-specific marker and an ideally suited drug target for PA.
Non-central nervous system hemangiopericytoma (HPC) and solitary fibrous tumor (SFT) are considered by pathologists as two variants of a single tumor entity now subsumed under the entity SFT. Recent ...detection of frequent
NAB2-STAT6
fusions in both, HPC and SFT, provided additional support for this view. On the other hand, current neuropathological practice still distinguishes between HPC and SFT. The present study set out to identify genes involved in the formation of meningeal HPC. We performed exome sequencing and detected the
NAB2-STAT6
fusion in DNA of 8/10 meningeal HPC thereby providing evidence of close relationship of these tumors with peripheral SFT. Due to the considerable effort required for exome sequencing, we sought to explore surrogate markers for the NAB2-STAT6 fusion protein. We adopted the Duolink proximity ligation assay and demonstrated the presence of NAB2-STAT6 fusion protein in 17/17 HPC and the absence in 15/15 meningiomas. More practical, presence of the NAB2-STAT6 fusion protein resulted in a strong nuclear signal in STAT6 immunohistochemistry. The nuclear reallocation of STAT6 was detected in 35/37 meningeal HPC and 25/25 meningeal SFT but not in 87 meningiomas representing the most important differential diagnosis. Tissues not harboring the NAB2-STAT6 fusion protein presented with nuclear expression of NAB2 and cytoplasmic expression of STAT6 proteins. In conclusion, we provide strong evidence for meningeal HPC and SFT to constitute variants of a single entity which is defined by
NAB2-STAT6
fusion. In addition, we demonstrate that this fusion can be rapidly detected by STAT6 immunohistochemistry which shows a consistent nuclear reallocation. This immunohistochemical assay may prove valuable for the differentiation of HPC and SFT from other mesenchymal neoplasms.
We present a tomographic model of radially anisotropic shear velocity variations in the Earth's mantle based on a new compilation of previously published data sets and a variable block ...parameterization, adapted to local raypath density. We employ ray‐theoretical sensitivity functions to relate surface wave and body wave data with radially anisotropic velocity perturbations. Our database includes surface wave phase delays from fundamental modes up to the sixth overtone, measured at periods between 25 and 350 s, as well as cross‐correlation traveltimes of major body wave phases. Before inversion, we apply crustal corrections using the crustal model CRUST2.0, and we account for azimuthal anisotropy in the upper mantle using ray‐theoretical corrections based on a global model of azimuthal anisotropy. While being well correlated with earlier models at long spatial wavelength, our preferred solution,
savani
, additionally delineates a number of previously unidentified structures due to its improved resolution in areas of dense coverage. This is because the density of the inverse grid ranges between 1.25° in well‐sampled and 5° in poorly sampled regions, allowing us to resolve regional structure better than it is typically the case in global
S
wave tomography. Our model highlights (i) a distinct ocean‐continent anisotropic signature in the uppermost mantle, (ii) an oceanic peak in above average
ξ
<1 which is shallower than in previous models and thus in better agreement with estimates of lithosphere thickness, and (iii) a long‐wavelength pattern of
ξ
<1 associated with the large low‐shear velocity provinces in the lowermost mantle.
Key Points
Variable resolution model of global radial shear wave anisotropy
Peak in ξ>1 under oceans in better agreement with LAB depths
ξ<1 near the large low–shear velocity provinces at the CMB
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