Astrocytomas are the most common type of brain tumors in children. Activated BRAF protein kinase mutations are characteristic of pediatric astrocytomas with KIAA1549-BRAF fusion genes typifying ...low-grade astrocytomas and ⱽ⁶⁰⁰ᴱBRAF alterations characterizing distinct or higher-grade tumors. Recently, BRAF-targeted therapies, such as vemurafenib, have shown great promise in treating V600E-dependent melanomas. Like ⱽ⁶⁰⁰ᴱBRAF, BRAF fusion kinases activate MAPK signaling and are sufficient for malignant transformation; however, here we characterized the distinct mechanisms of action of KIAA1549-BRAF and its differential responsiveness to PLX4720, a first-generation BRAF inhibitor and research analog of vemurafenib. We found that in cells expressing KIAA1549-BRAF, the fusion kinase functions as a homodimer that is resistant to PLX4720 and accordingly is associated with CRAF-independent paradoxical activation of MAPK signaling. Mutagenesis studies demonstrated that KIAA1549-BRAF fusion-mediated signaling is diminished with disruption of the BRAF kinase dimer interface. In addition, the KIAA1549-BRAF fusion displays increased binding affinity to kinase suppressor of RAS (KSR), an RAF relative recently demonstrated to facilitate MEK phosphorylation by BRAF. Despite its resistance to PLX4720, the KIAA1549-BRAF fusion is responsive to a second-generation selective BRAF inhibitor that, unlike vemurafenib, does not induce activation of wild-type BRAF. Our data support the development of targeted treatment paradigms for BRAF-altered pediatric astrocytomas and also demonstrate that therapies must be tailored to the specific mutational context and distinct mechanisms of action of the mutant kinase.
T cells made with messenger RNA (mRNA) encoding chimeric antigen receptor (CAR) offer a safe alternative to those transduced with viral CARs by mitigating the side effects of constitutively active T ...cells. Previous studies have shown that mRNA CAR T cells are transiently effective but lack persistence and potency across tumor types. It was hypothesized that the efficacy of mRNA CARs could be improved by utilizing recent advancements in RNA technology, such as incorporating a modified nucleoside, 1-methylpseudouridine, into the mRNA and applying a novel purification method using RNase III to eliminate dsRNA contaminants. T cells electroporated with nucleoside-modified and purified mRNA encoding CD19 CAR showed an initial twofold increase in CAR surface expression, as well as a twofold improvement in cytotoxic killing of leukemia cells that persisted up to 5 days. T cells generated with nucleoside-modified and purified CAR mRNA also showed reduced expression of checkpoint regulators and a differential pattern of genetic activation compared to those made with conventional mRNA. In vivo studies using a leukemia mouse model revealed that the most robust 100-fold suppression of leukemic burden was achieved using T cells electroporated with purified mRNAs, regardless of their nucleoside modification. The results provide a novel approach to generate mRNA for clinical trials, and poise mRNA CAR T cells for increased efficacy during testing as new CAR targets emerge.
Summary
Purpose: The role of sharps and spikes, interictal epileptiform discharges (IEDs), in guiding epilepsy surgery in children remains controversial, particularly with intracranial ...electroencephalography (IEEG). Although ictal recording is the mainstay of localizing epileptic networks for surgical resection, current practice dictates removing regions generating frequent IEDs if they are near the ictal onset zone. Indeed, past studies suggest an inconsistent relationship between IED and seizure‐onset location, although these studies were based upon relatively short EEG epochs.
Methods: We employ a previously validated, computerized spike detector to measure and localize IED activity over prolonged, representative segments of IEEG recorded from 19 children with intractable, mostly extratemporal lobe epilepsy. Approximately 8 h of IEEG, randomly selected 30‐min segments of continuous interictal IEEG per patient, were analyzed over all intracranial electrode contacts.
Results: When spike frequency was averaged over the 16‐time segments, electrodes with the highest mean spike frequency were found to be within the seizure‐onset region in 11 of 19 patients. There was significant variability between individual 30‐min segments in these patients, indicating that large statistical samples of interictal activity were required for improved localization. Low‐voltage fast EEG at seizure onset was the only clinical factor predicting IED localization to the seizure‐onset region.
Conclusions: Our data suggest that automated IED detection over multiple representative samples of IEEG may be of utility in planning epilepsy surgery for children with intractable epilepsy. Further research is required to better determine which patients may benefit from this technique a priori.
In the present study, DNA from 27 grade I and grade II pediatric gliomas, including ganglioglioma, desmoplastic infantile ganglioglioma, dysembryoplastic neuroepithelial tumor, and pleomorphic ...xanthoastrocytoma was analyzed using the Illumina 610K Beadchip SNP-based oligonucleotide array. Several consistent abnormalities, including gain of chromosome 7 and loss of 9p21 were observed. Based on our previous studies, in which we demonstrated BRAF mutations in 3 gangliogliomas, 31 tumors were screened for activating mutations in exons 11 and 15 of the BRAF oncogene or a KIAA1549-BRAF fusion product. There were no cases with a KIAA1549-BRAF fusion. A BRAF V600E mutation was detected in 14 of 31 tumors, which was not correlated with any consistent pattern of aberrations detected by the SNP array analysis. Tumors were also screened for mutations in codon 132 in exon 4 of IDH1, exons 2 and 3 of KRAS, and exons 2-9 of TP53. No mutations in KRAS or TP53 were identified in any of the samples, and there was only 1 IDH1 R132H mutation detected among the sample set. BRAF mutations constitute a major genetic alteration in this histologic group of pediatric brain tumors and may serve as a molecular target for biologically based inhibitors.
Abstract
Dysembryoplastic neuroepithelial tumors (DNT) lacking key diagnostic criteria are challenging to diagnose and sometimes fall into the broader category of mixed neuronal-glial tumors (MNGT) ...or the recently described polymorphous low-grade neuroepithelial tumor of the young (PLNTY). We examined 41 patients with DNT, MNGT, or PLNTY for histologic features, genomic findings, and progression-free survival (PFS). Genomic analysis included sequence and copy number variants and RNA-sequencing. Classic DNT (n = 26) was compared with those with diffuse growth without cortical nodules (n = 15), 6 of which exhibited impressive CD34 staining classifying them as PLNTY. Genomic analysis was complete in 33, with sequence alterations recurrently identified in BRAF, FGFR1, NF1, and PDGFRA, as well as 7 fusion genes involving FGFR2, FGFR1, NTRK2, and BRAF. Genetic alterations did not distinguish between MNGTs, DNTs, or PLNTYs; however, FGFR1 alterations were confined to DNT, and PLNTYs contained BRAF V600E or FGFR2 fusion genes. Analysis of PFS showed no significant difference by histology or genetic alteration; however, numbers were small and follow-up time short. Further molecular characterization of a PLNTY-related gene fusion, FGFR2-CTNNA3, demonstrated oncogenic potential via MAPK/PI3K/mTOR pathway activation. Overall, DNT-MNGT spectrum tumors exhibit diverse genomic alterations, with more than half (19/33) leading to MAPK/PI3K pathway alterations.
Despite advancements in molecular and histopathologic characterization of pediatric low-grade gliomas (pLGGs), there remains significant phenotypic heterogeneity among tumors with similar ...categorizations. We hypothesized that an unsupervised machine learning approach based on radiomic features may reveal distinct pLGG imaging subtypes.
Multi-parametric MR images (T1 pre- and post-contrast, T2, and T2 FLAIR) from 157 patients with pLGGs were collected and 881 quantitative radiomic features were extracted from tumorous region. Clustering was performed using K-means after applying principal component analysis (PCA) for feature dimensionality reduction. Molecular and demographic data was obtained from the PedCBioportal and compared between imaging subtypes.
K-means identified three distinct imaging-based subtypes. Subtypes differed in mutational frequencies of BRAF (p < 0.05) as well as the gene expression of BRAF (p<0.05). It was also found that age (p < 0.05), tumor location (p < 0.01), and tumor histology (p < 0.0001) differed significantly between the imaging subtypes.
In this exploratory work, it was found that clustering of pLGGs based on radiomic features identifies distinct, imaging-based subtypes that correlate with important molecular markers and demographic details. This finding supports the notion that incorporation of radiomic data could augment our ability to better characterize pLGGs.
Medulloblastoma is a highly heterogeneous pediatric brain tumor with five molecular subtypes, Sonic Hedgehog TP53-mutant, Sonic Hedgehog TP53-wildtype, WNT, Group 3, and Group 4, defined by the World ...Health Organization. The current mechanism for classification into these molecular subtypes is through the use of immunostaining, methylation, and/or genetics. We surveyed the literature and identified a number of RNA-Seq and microarray datasets in order to develop, train, test, and validate a robust classifier to identify medulloblastoma molecular subtypes through the use of transcriptomic profiling data. We have developed a GPL-3 licensed R package and a Shiny Application to enable users to quickly and robustly classify medulloblastoma samples using transcriptomic data. The classifier utilizes a large composite microarray dataset (15 individual datasets), an individual microarray study, and an RNA-Seq dataset, using gene ratios instead of gene expression measures as features for the model. Discriminating features were identified using the limma R package and samples were classified using an unweighted mean of normalized scores. We utilized two training datasets and applied the classifier in 15 separate datasets. We observed a minimum accuracy of 85.71% in the smallest dataset and a maximum of 100% accuracy in four datasets with an overall median accuracy of 97.8% across the 15 datasets, with the majority of misclassification occurring between the heterogeneous Group 3 and Group 4 subtypes. We anticipate this medulloblastoma transcriptomic subtype classifier will be broadly applicable to the cancer research and clinical communities.
Detecting elevated intracranial pressure in children with subacute conditions, such as craniosynostosis or tumor, may enable timely intervention and prevent neurocognitive impairment, but ...conventional techniques are invasive and often equivocal. Elevated intracranial pressure leads to structural changes in the peripapillary retina. Spectral-domain (SD) optical coherence tomography (OCT) can noninvasively quantify retinal layers to a micron-level resolution.
To evaluate whether retinal measurements from OCT can serve as an effective surrogate for invasive intracranial pressure measurement.
This cross-sectional study included patients undergoing procedures at the Children's Hospital of Philadelphia from September 2014 to June 2015. Three groups of patients (n = 79) were prospectively enrolled from the Craniofacial Surgery clinic including patients with craniosynostosis (n = 40). The positive control cohort consisted of patients with hydrocephalus and suspected intracranial hypertension (n = 5), and the negative control cohort consisted of otherwise healthy patients undergoing a minor procedure (n = 34).
Spectral-domain OCT was performed preoperatively in all cohorts. Children with cranial pathology, but not negative control patients, underwent direct intraoperative intracranial pressure measurement. The primary outcome was the association between peripapillary retinal OCT parameters and directly measured elevated intracranial pressure.
The mean (SD) age was 34.6 (45.2) months in the craniosynostosis cohort (33% female), 48.9 (83.8) months in the hydrocephalus and suspected intracranial hypertension cohort (60% female), and 59.7 (64.4) months in the healthy cohort (47% female). Intracranial pressure correlated with maximal retinal nerve fiber layer thickness (r = 0.60, P ≤ .001), maximal retinal thickness (r = 0.53, P ≤ .001), and maximal anterior retinal projection (r = 0.53, P = .003). Using cut points derived from the negative control patients, OCT parameters yielded 89% sensitivity (95% CI, 69%-97%) and 62% specificity (95% CI, 41%-79%) for detecting elevated intracranial pressure. The SD-OCT measures had high intereye agreement (intraclass correlation, 0.83-0.93) and high intragrader and intergrader agreement (intraclass correlation ≥0.94). Conventional clinical signs had low sensitivity (11%-42%) for detecting intracranial hypertension.
Noninvasive quantitative measures of the peripapillary retinal structure by SD-OCT were correlated with invasively measured intracranial pressure. Optical coherence tomographic parameters showed promise as surrogate, noninvasive measures of intracranial pressure, outperforming other conventional clinical measures. Spectral-domain OCT of the peripapillary region has the potential to advance current treatment paradigms for elevated intracranial pressure in children.
•Analysis of biorepository of craniopharyngioma data shows robust genomic sequencing resources, but lack of information on functional and quality of life (QoL) outcomes.•Patient and family survey ...demonstrates treatment and outcome priorities at time of initial diagnosis and recurrence.•Patients and families suggest prioritization of more effective prospective treatments that may improve quality-of-life even if increased risk of recurrence.
Craniopharyngioma is a rare, low-grade tumor located in the suprasellar region of the brain, near critical structures like the pituitary gland. Here, we concurrently investigate the status of clinical and genomic data in a retrospective craniopharyngioma cohort and survey-based data to better understand patient-relevant outcomes associated with existing therapies and provide a foundation to inform new treatment strategies.
Clinical, genomic, and outcome data for a retrospective cohort of patients with craniopharyngioma were collected and reviewed through the Children's Brain Tumor Network (CBTN) database. An anonymous survey was distributed to patients and families with a diagnosis of craniopharyngioma to understand their experiences throughout diagnosis and treatment.
The CBTN repository revealed a large proportion of patients (40 – 70%) with specimens that are available for sequencing but lacked relevant quality of life (QoL) and functional outcomes. Frequencies of reported patient comorbidities ranged from 20–35%, which is significantly lower than historically reported. Survey results from 159 patients/families identified differences in treatment considerations at time of diagnosis versus time of recurrence. In retrospective review, patients and families identified preference for therapy that would improve QoL, rather than decrease risk of recurrence (mean 3.9 vs. 4.4 of 5) and identified endocrine issues as having the greatest impact on patients’ lives.
This work highlights the importance of prospective collection of QoL and functional metrics alongside robust clinical and molecular correlates in individuals with craniopharyngioma. Such comprehensive measures will facilitate biologically relevant therapeutic strategies that also prioritize patient needs.
The purposes of this study were to determine the incidence of neurovascular events as late complications in pediatric patients with brain tumor and to evaluate radiation as a risk factor.
Patients ...were ascertained using the tumor database of a pediatric tertiary care center. Included patients had a primary brain tumor, age birth to 21 years, initial treatment January 1, 1993, to December 31, 2002, and at least 2 visits with neuro-oncology. Radiation exposure included: whole brain, whole brain plus a focal boost, or focal brain. The primary outcome was stroke or transient ischemic attack.
Of 431 subjects, 14 had 19 events of stroke or transient ischemic attack over a median follow-up of 6.3 years. The incidence rate was 548/100 000 person-years. Overall, 61.5% of subjects received radiation, including 13 of 14 subjects with events. Median time from first radiation to first event was 4.9 years. The stroke/transient ischemic attack hazard ratio for any brain irradiation was 8.0 (95% CI, 1.05-62; P=0.045); for the circle of Willis, radiation was 9.0 (95% CI, 1.2-70; P=0.035); and for focal noncircle of Willis, radiation was 3.4 (95% CI, 0.21-55; P=0.38).
The incidence of neurovascular events in this population is 100-fold higher than in the general pediatric population and cranial irradiation is an important risk factor. By defining the incidence of this late effect, physicians are better able to counsel parents regarding treatment, monitor patients at risk, and target a population for primary stroke prevention in future studies.
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