Phillip B. Storm

The genetic landscape of the childhood cancer medulloblastoma

Genomic analysis of a childhood cancer reveals markedly fewer mutations than what is typically seen in adult cancers. Medulloblastoma (MB) is the most common malignant brain tumor of children. To identify the genetic alterations in this tumor type, we searched for copy number alterations using high-density microarrays and sequenced all known protein-coding genes and microRNA genes using Sanger sequencing in a set of 22 MBs. We found that, on average, each tumor had 11 gene alterations, fewer by a factor of 5 to 10 than in the adult solid tumors that have been sequenced to date. In addition to alterations in the Hedgehog and Wnt pathways, our analysis led to the discovery of genes not previously known to be altered in MBs. Most notably, inactivating mutations of the histone-lysine N-methyltransferase genes MLL2 or MLL3 were identified in 16% of MB patients. These results demonstrate key differences between the genetic landscapes of adult and childhood cancers, highlight dysregulation of developmental pathways as an important mechanism underlying MBs, and identify a role for a specific type of histone methylation in human tumorigenesis.

Activating mutations in BRAF characterize a spectrum of pediatric low-grade gliomas

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.

Duplication of 7q34 in Pediatric Low-Grade Astrocytomas Detected by High-Density Single-Nucleotide Polymorphism-Based Genotype Arrays Results in a Novel BRAF Fusion Gene

In the present study, DNA from 28 pediatric low‐grade astrocytomas was analyzed using Illumina HumanHap550K single‐nucleotide polymorphism oligonucleotide arrays. A novel duplication in chromosome band 7q34 was identified in 17 of 22 juvenile pilocytic astrocytomas and three of six fibrillary astrocytomas. The 7q34 duplication spans 2.6 Mb of genomic sequence and contains approximately 20 genes, including two candidate tumor genes, HIPK2 and BRAF. There were no abnormalities in HIPK2, and analysis of two mutation hot‐spots in BRAF revealed a V600E mutation in only one tumor without the duplication. Fluorescence in situ hybridization confirmed the 7q34 copy number change and was suggestive of a tandem duplication. Reverse transcription polymerase chain reaction‐based sequencing revealed a fusion product between KIAA1549 and BRAF. The predicted fusion product includes the BRAF kinase domain and lacks the auto‐inhibitory N‐terminus. Western blot analysis revealed phosphorylated mitogen‐activated protein kinase (MAPK) protein in tumors with the duplication, consistent with BRAF‐induced activation of the pathway. Further studies are required to determine the role of this fusion gene in downstream MAPK signaling and its role in development of pediatric low‐grade astrocytomas.

Exome sequencing identifies BRAF mutations in papillary craniopharyngiomas

Craniopharyngiomas are epithelial tumors that typically arise in the suprasellar region of the brain. Patients experience substantial clinical sequelae from both extension of the tumors and therapeutic interventions that damage the optic chiasm, the pituitary stalk and the hypothalamic area. Using whole-exome sequencing, we identified mutations in CTNNB1 (β-catenin) in nearly all adamantinomatous craniopharyngiomas examined (11/12, 92%) and recurrent mutations in BRAF (resulting in p.Val600Glu) in all papillary craniopharyngiomas (3/3, 100%). Targeted genotyping revealed BRAF p.Val600Glu in 95% of papillary craniopharyngiomas (36 of 39 tumors) and mutation of CTNNB1 in 96% of adamantinomatous craniopharyngiomas (51 of 53 tumors). The CTNNB1 and BRAF mutations were clonal in each tumor subtype, and we detected no other recurrent mutations or genomic aberrations in either subtype. Adamantinomatous and papillary craniopharyngiomas harbor mutations that are mutually exclusive and clonal. These findings have important implications for the diagnosis and treatment of these neoplasms.

Paradoxical activation and RAF inhibitor resistance of BRAF protein kinase fusions characterizing pediatric astrocytomas

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.

Polymer Delivery of Camptothecin against 9L Gliosarcoma: Release, Distribution, and Efficacy

Camptothecin is a potent antineoplastic agent that has shown efficacy against multiple tumor lines in vitro; unfortunately, systemic toxicity has limited its in vivo efficacy. This is the first study to investigate the release, biodistribution, and efficacy of camptothecin from a biodegradable polyanhydride polymer. Tritiated camptothecin was incorporated into biodegradable polymers that were implanted intracranially in 16 male Fischer 344 rats and the animals were followed up to 21 days post-implant. A concentration of 11–45 μg of camptothecin-sodium/mg brain tissue was within a 3 mm radius of the polymer disc, with levels of 0.1 μg at the outermost margin of the rat brain, 7 mm from the site of implantation. These tissue concentrations are within the therapeutic ranges for human and rat glioma lines tested against camptothecin-sodium in vitro. The in vivo efficacy of camptothecin-sodium was evaluated with male Fischer 344 rats implanted intracranially with 9L gliosarcoma and compared with the efficacy of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). The animals were divided into four groups. Group 1 (control) had a median survival of 17 days. Group 2 (3.8% BCNU polymer) had a median survival of 23 days (P=0.006). Group 3 (20% camptothecin polymer) had a median survival of 25 days (P=0.023). Group 4 (50% camptothecin polymer) had a median survival of 69 days (P<0.001). Drug loadings of 20% and 50% camptothecin released intact camptothecin for up to 1000 h in vitro. We conclude that the biodegradable polymer p(CPP : SA) releases camptothecin-sodium, produces tumoricidal tissue levels, results in little or no systemic toxicity, and prolongs survival in a rat glioma model.

Predictors of Outcome in Childhood Intracerebral Hemorrhage A Prospective Consecutive Cohort Study

Background and Purpose— The purposes of this study were to describe features of children with intracerebral hemorrhage (ICH) and to determine predictors of short-term outcome in a single-center prospective cohort study. Methods— A single-center prospective consecutive cohort study was conducted of spontaneous ICH in children aged 1 to 18 years from January 2006 to June 2008. Exclusion criteria were inciting trauma; intracranial tumor; isolated epidural, subdural, intraventricular, or subarachnoid hemorrhage; hemorrhagic transformation of ischemic stroke; and cerebral sinovenous thrombosis. Hospitalization records were abstracted. Follow-up assessments included outcome scores using the Pediatric Stroke Outcome Measure and Kings Outcome Scale for Childhood Head Injury. ICH volumes and total brain volumes were measured by manual tracing. Results— Twenty-two patients, median age 10.3 years (range, 4.2 to 16.6 years), had presenting symptoms of headache in 77%, focal deficits 50%, altered mental status 50%, and seizures 41%. Vascular malformations caused hemorrhage in 91%. Surgical treatment (hematoma evacuation, lesion embolization or excision) was performed during acute hospitalization in 50%. One patient died acutely. At a median follow-up of 3.5 months (range, 0.3 to 7.5 months), 71% of survivors had neurological deficits; 55% had clinically significant disability. Outcome based on Pediatric Stroke Outcome Measure and Kings Outcome Scale for Childhood Head Injury scores was worse in patients with ICH volume >2% of total brain volume (P=0.023) and altered mental status at presentation (P=0.005). Conclusions— Spontaneous childhood ICH was due mostly to vascular malformations. Acute surgical intervention was commonly performed. Although death was rare, 71% of survivors had persisting neurological deficits. Larger ICH volume and altered mental status predicted clinically significant disability.

MYB-QKI rearrangements in angiocentric glioma drive tumorigenicity through a tripartite mechanism.

Angiocentric gliomas are pediatric low-grade gliomas (PLGGs) without known recurrent genetic drivers. We performed genomic analysis of new and published data from 249 PLGGs, including 19 angiocentric gliomas. We identified MYB-QKI fusions as a specific and single candidate driver event in angiocentric gliomas. In vitro and in vivo functional studies show that MYB-QKI rearrangements promote tumorigenesis through three mechanisms: MYB activation by truncation, enhancer translocation driving aberrant MYB-QKI expression and hemizygous loss of the tumor suppressor QKI. To our knowledge, this represents the first example of a single driver rearrangement simultaneously transforming cells via three genetic and epigenetic mechanisms in a tumor.

Inositol polyphosphate multikinase is a physiologic PI3-kinase that activates Akt/PKB

The second messenger phosphatidylinositol (3,4,5)-trisphosphate (PIP3), formed by the p110 family of PI3-kinases, promotes cellular growth, proliferation, and survival, in large part by activating the protein kinase Akt/PKB. We show that inositol polyphosphate multikinase (IPMK) physiologically generates PIP3 as well as water soluble inositol phosphates. IPMK deletion reduces growth factor-elicited Akt signaling and cell proliferation caused uniquely by loss of its PI3-kinase activity. Inhibition of p110 PI3-kinases by wortmannin prevents IPMK phosphorylation and activation. Thus, growth factor stimulation of Akt signaling involves PIP3 generation through the sequential activations of the p110 PI3-kinases and IPMK. As inositol phosphates inhibit Akt signaling, IPMK appears to act as a molecular switch, inhibiting or stimulating Akt via its inositol phosphate kinase or PI3-kinase activities, respectively. Drugs regulating IPMK may have therapeutic relevance in influencing cell proliferation.

Cranial Irradiation Increases Risk of Stroke in Pediatric Brain Tumor Survivors

Background and Purpose— 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. Methods— 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. Results— 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). Conclusions— 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.