Jeffrey R. Leonard

Recurrent somatic mutations in ACVR1 in pediatric midline high-grade astrocytoma

Pediatric midline high-grade astrocytomas (mHGAs) are incurable with few treatment targets identified. Most tumors harbor mutations encoding p.Lys27Met in histone H3 variants. In 40 treatment-naive mHGAs, 39 analyzed by whole-exome sequencing, we find additional somatic mutations specific to tumor location. Gain-of-function mutations in ACVR1 occur in tumors of the pons in conjunction with histone H3.1 p.Lys27Met substitution, whereas FGFR1 mutations or fusions occur in thalamic tumors associated with histone H3.3 p.Lys27Met substitution. Hyperactivation of the bone morphogenetic protein (BMP)-ACVR1 developmental pathway in mHGAs harboring ACVR1 mutations led to increased levels of phosphorylated SMAD1, SMAD5 and SMAD8 and upregulation of BMP downstream early-response genes in tumor cells. Global DNA methylation profiles were significantly associated with the p.Lys27Met alteration, regardless of the mutant histone H3 variant and irrespective of tumor location, supporting the role of this substitution in driving the epigenetic phenotype. This work considerably expands the number of potential treatment targets and further justifies pretreatment biopsy in pediatric mHGA as a means to orient therapeutic efforts in this disease.

Subgroup-Specific Prognostic Implications of TP53 Mutation in Medulloblastoma

PURPOSE Reports detailing the prognostic impact of TP53 mutations in medulloblastoma offer conflicting conclusions. We resolve this issue through the inclusion of molecular subgroup profiles. PATIENTS AND METHODS We determined subgroup affiliation, TP53 mutation status, and clinical outcome in a discovery cohort of 397 medulloblastomas. We subsequently validated our results on an independent cohort of 156 medulloblastomas. RESULTS TP53 mutations are enriched in wingless (WNT; 16%) and sonic hedgehog (SHH; 21%) medulloblastomas and are virtually absent in subgroups 3 and 4 tumors (P < .001). Patients with SHH/TP53 mutant tumors are almost exclusively between ages 5 and 18 years, dramatically different from the general SHH distribution (P < .001). Children with SHH/TP53 mutant tumors harbor 56% germline TP53 mutations, which are not observed in children with WNT/TP53 mutant tumors. Five-year overall survival (OS; ± SE) was 41% ± 9% and 81% ± 5% for patients with SHH medulloblastomas with and without TP53 mutations, respectively (P < .001). Furthermore, TP53 mutations accounted for 72% of deaths in children older than 5 years with SHH medulloblastomas. In contrast, 5-year OS rates were 90% ± 9% and 97% ± 3% for patients with WNT tumors with and without TP53 mutations (P = .21). Multivariate analysis revealed that TP53 status was the most important risk factor for SHH medulloblastoma. Survival rates in the validation cohort mimicked the discovery results, revealing that poor survival of TP53 mutations is restricted to patients with SHH medulloblastomas (P = .012) and not WNT tumors. CONCLUSION Subgroup-specific analysis reconciles prior conflicting publications and confirms that TP53 mutations are enriched among SHH medulloblastomas, in which they portend poor outcome and account for a large proportion of treatment failures in these patients.

Distinct Genetic Signatures among Pilocytic Astrocytomas Relate to Their Brain Region Origin

Pilocytic astrocytomas (PAs) are the most common glioma in children. Whereas many PAs are slow-growing or clinically indolent, others exhibit more aggressive features with tumor recurrence and death. To identify genetic signatures that might predict PA clinical behavior, we did gene expression profiling on 41 primary PAs arising sporadically and in patients with neurofibromatosis type 1 (NF1). Whereas no expression signature was found that could discriminate clinically aggressive or recurrent tumors from more indolent cases, PAs arising in patients with NF1 did exhibit a unique gene expression pattern. In addition, we identified a gene expression signature that stratified PAs by location (supratentorial versus infratentorial). Lastly, we also identified a gene expression pattern common to PAs and normal mouse astrocytes and neural stem cells from these distinct brain regions as well as a gene expression pattern shared between PAs and another human glial tumor (ependymoma) arising supratentorially compared with those originating in the posterior fossa. These results suggest that glial tumors share an intrinsic, lineage-specific molecular signature that reflects the brain region in which their nonmalignant predecessors originated.

Cytogenetic Prognostication Within Medulloblastoma Subgroups

PURPOSE Medulloblastoma comprises four distinct molecular subgroups: WNT, SHH, Group 3, and Group 4. Current medulloblastoma protocols stratify patients based on clinical features: patient age, metastatic stage, extent of resection, and histologic variant. Stark prognostic and genetic differences among the four subgroups suggest that subgroup-specific molecular biomarkers could improve patient prognostication. PATIENTS AND METHODS Molecular biomarkers were identified from a discovery set of 673 medulloblastomas from 43 cities around the world. Combined risk stratification models were designed based on clinical and cytogenetic biomarkers identified by multivariable Cox proportional hazards analyses. Identified biomarkers were tested using fluorescent in situ hybridization (FISH) on a nonoverlapping medulloblastoma tissue microarray (n = 453), with subsequent validation of the risk stratification models. RESULTS Subgroup information improves the predictive accuracy of a multivariable survival model compared with clinical biomarkers alone. Most previously published cytogenetic biomarkers are only prognostic within a single medulloblastoma subgroup. Profiling six FISH biomarkers (GLI2, MYC, chromosome 11 [chr11], chr14, 17p, and 17q) on formalin-fixed paraffin-embedded tissues, we can reliably and reproducibly identify very low-risk and very high-risk patients within SHH, Group 3, and Group 4 medulloblastomas. CONCLUSION Combining subgroup and cytogenetic biomarkers with established clinical biomarkers substantially improves patient prognostication, even in the context of heterogeneous clinical therapies. The prognostic significance of most molecular biomarkers is restricted to a specific subgroup. We have identified a small panel of cytogenetic biomarkers that reliably identifies very high-risk and very low-risk groups of patients, making it an excellent tool for selecting patients for therapy intensification and therapy de-escalation in future clinical trials.

Gliomas in neurofibromatosis type 1: a clinicopathologic study of 100 patients.

There are few pathologic studies of gliomas in patients with neurofibromatosis type 1. We analyzed clinical and pathologic features of gliomas from 100 neurofibromatosis type 1 patients (57 men; 43 women). The median age at tumor diagnosis was 13 years (range, 4 months to 68 years). Most tumors were typical pilocytic astrocytoma (PA) (49%) or diffusely infiltrating astrocytoma (DA) (27%) that included World Health Organization Grades II (5%), III (15%), and IV (7%); others were designated as low-grade astrocytoma, subtype indeterminate (LGSI; 17%). Two pilomyxoid astrocytomas, 1 desmoplastic infantile ganglioglioma and 1 conventional ganglioglioma, were also identified. The tumors in 24 cases arose in the optic pathways and included PA (n = 14), LGSI (n = 4), DA (n = 4), pilomyxoid astrocytoma (n = 1), and ganglioglioma (n = 1). The prognoses of the PA and LGSI gliomas overall were generally favorable; there were no survival differences between PA and LGSI groups based on site, tumor size, mitotic activity, or MIB-1 labeling index. In the combined PA and LGSI group, age younger than 10 years and gross total resection were associated with an increased overall survival rate (p = 0.047 and 0.002, respectively). Compared with the combined group (PA + LGSI), patients with DA at all sites had decreased overall and recurrence-free survival times (p < 0.001 and p = 0.003, respectively). This study emphasizes the wide histologic spectrum of gliomas that occur in patients with neurofibromatosis type 1. Classic PA and LGSI are the most common, and most have favorable prognoses. By contrast, DAs are more aggressive, similar to those that arise sporadically.

High-resolution, dual-platform aCGH analysis reveals frequent HIPK2 amplification and increased expression in pilocytic astrocytomas

Pilocytic astrocytomas (PAs, WHO grade I) are the most common brain tumors in the pediatric and adolescent population, accounting for approximately one-fifth of central nervous system tumors. Because few consistent molecular alterations have been identified in PAs compared to higher grade gliomas, we performed array comparative genomic hybridization using two independent commercial array platforms. Although whole chromosomal gains and losses were not observed, a 1-Mb amplified region of 7q34 was detected in multiple patient samples using both array platforms. Copy-number gain was confirmed in an independent tumor sample set by quantitative PCR, and this amplification was correlated to both increased mRNA and protein expression of HIPK2, a homeobox-interacting protein kinase associated with malignancy, contained within this locus. Furthermore, overexpression of wild-type HIPK2, but not a kinase-inactive mutant, in a glioma cell line conferred a growth advantage in vitro. Collectively, these results illustrate the power and necessity of implementing high-resolution, multiple-platform genomic analyses to discover small and subtle, but functionally significant, genomic alterations associated with low-grade tumor formation and growth.

Intertumoral Heterogeneity within Medulloblastoma Subgroups

While molecular subgrouping has revolutionized medulloblastoma classification, the extent of heterogeneity within subgroups is unknown. Similarity network fusion (SNF) applied to genome-wide DNA methylation and gene expression data across 763 primary samples identifies very homogeneous clusters of patients, supporting the presence of medulloblastoma subtypes. After integration of somatic copy-number alterations, and clinical features specific to each cluster, we identify 12 different subtypes of medulloblastoma. Integrative analysis using SNF further delineates group 3 from group 4 medulloblastoma, which is not as readily apparent through analyses of individual data types. Two clear subtypes of infants with Sonic Hedgehog medulloblastoma with disparate outcomes and biology are identified. Medulloblastoma subtypes identified through integrative clustering have important implications for stratification of future clinical trials.

Long-term outcome in children treated for craniopharyngioma with and without radiotherapy

OBJECT The authors report the results of surgery alone or in combination with radiotherapy in the management of craniopharyngiomas in children. METHODS The authors retrospectively reviewed the outcomes in 31 patients treated for craniopharyngiomas at the Department of Radiation Oncology at Washington University in St. Louis and the St. Louis Childrens Hospital. The median age at diagnosis was 8.1 years (range 1.1-21 years). Fourteen patients underwent gross-total resection (GTR) with observation, and 6 patients underwent subtotal resection (STR) with observation. Ten patients underwent STR or cyst aspiration followed by external-beam radiotherapy, and 1 patient underwent cyst aspiration followed by intracystic 32P installation. RESULTS The median follow-up for all surviving patients was 78.2 months. Overall survival and local control rates at 10 years were 96 and 58%, respectively. One patient died of the disease, and 12 patients had subsequent recurrences. Of those with recurrences, 6 patients had undergone initial STR with observation and 6 had been treated with GTR and observation. The median time to progression was 17.9 months in the patients who underwent limited resection, and 55 months for those who underwent GTR. There were no recurrences in the patients who received radiotherapy at the time of initial diagnosis. CONCLUSIONS Radiotherapy delivered as part of the initial management of craniopharyngiomas in children or at the time of recurrence provides effective local control.

19F- and Fluorescently Labeled Micelles as Nanoscopic Assemblies for Chemotherapeutic Delivery

Micelles from amphiphilic star-block copolymers, having a hydrophobic hyperbranched core and amphiphilic fluoropolymer arms, were constructed as drug delivery agent assemblies. A series of polymer structures was constructed from consecutive copolymerizations of 4-chloromethylstyrene with dodecyl acrylate and then 1,1,1- trifluoroethyl methacrylate with tert-butyl acrylate, followed by acidolysis to release the hydrophilic acrylic acid residues. These structures were labeled with cascade blue as a fluorescence reporter. The series of materials differed primarily in the ratio of 1,1,1-trifluoroethyl methacrylate to acrylic acid units, to give differences in fluorine loading and hydrophobicity/hydrophilicity balance. Doxorubicin (DOX) was used as a therapeutic to study the loading, release, and cytotoxicity of these micellar constructs on an U87-MG-EGFRvIII-CBR cell line. The micelles, with TEM-measured diameters ranging 5-9 nm and DLS-measured hydrodynamic diameters 20-30 nm, had loading capacities of ca. 4 wt % of DOX. The DOX-loaded micelles exhibited potent cytotoxicity with cell viabilities of 60-25% at 1.0 microg/mL effective DOX concentrations, depending upon the polymer composition, as determined by MTT assays. These cell viability values are comparable to that of free DOX, suggesting an effective release of the cargo and delivery to the cell nuclei, which was further confirmed by fluorescence microscopy of the cells. 19F-NMR spectroscopy indicated a partial degradation of the surface-available trifluoroethyl ester linkages of the micelles, which may have accelerated the release of DOX. 19F-NMR spectroscopy was also employed to confirm and to quantify the cell uptake of the micelles. These dual fluorescent- and 19F-labeled and chemically functional micelles may be used potentially in a variety of applications, such as cell labeling, imaging, and therapeutic delivery.

Factors Associated With Cervical Spine Injury in Children After Blunt Trauma

STUDY OBJECTIVE Cervical spine injuries in children are rare. However, immobilization and imaging for potential cervical spine injury after trauma are common and are associated with adverse effects. Risk factors for cervical spine injury have been developed to safely limit immobilization and radiography in adults, but not in children. The purpose of our study is to identify risk factors associated with cervical spine injury in children after blunt trauma. METHODS We conducted a case-control study of children younger than 16 years, presenting after blunt trauma, and who received cervical spine radiographs at 17 hospitals in the Pediatric Emergency Care Applied Research Network (PECARN) between January 2000 and December 2004. Cases were children with cervical spine injury. We created 3 control groups of children free of cervical spine injury: (1) random controls, (2) age and mechanism of injury-matched controls, and (3) for cases receiving out-of-hospital emergency medical services (EMS), age-matched controls who also received EMS care. We abstracted data from 3 sources: PECARN hospital, referring hospital, and out-of-hospital patient records. We performed multiple logistic regression analyses to identify predictors of cervical spine injury and calculated the models sensitivity and specificity. RESULTS We reviewed 540 records of children with cervical spine injury and 1,060, 1,012, and 702 random, mechanism of injury, and EMS controls, respectively. In the analysis using random controls, we identified 8 factors associated with cervical spine injury: altered mental status, focal neurologic findings, neck pain, torticollis, substantial torso injury, conditions predisposing to cervical spine injury, diving, and high-risk motor vehicle crash. Having 1 or more factors was 98% (95% confidence interval 96% to 99%) sensitive and 26% (95% confidence interval 23% to 29%) specific for cervical spine injury. We identified similar risk factors in the other analyses. CONCLUSION We identified an 8-variable model for cervical spine injury in children after blunt trauma that warrants prospective refinement and validation.