Combining immunotherapy with high-dose radiation therapy (HDRT) significantly inhibits tumor growth in a syngeneic mouse model of high-risk neuroblastoma.

Purpose: The mortality in patients with MYCN-amplified high-risk neuroblastoma remains greater than 50% despite advances in multimodal therapy. Novel therapies are urgently needed that requires preclinical evaluation in appropriate mice models. Combinatorial treatment with high-dose radiotherapy (HDRT) and immunotherapy has emerged as an effective treatment option in a variety of cancers. Current models of neuroblastoma do not recapitulate the anatomic and immune environment in which multimodal therapies can be effectively tested, and there is a need for an appropriate syngeneic neuroblastoma mice model to study interaction of immunotherapy with host immune cells. Here, we develop a novel syngeneic mouse model of MYCN-amplified neuroblastoma and report the relevance and opportunities of this model to study radiotherapy and immunotherapy. Materials and methods: A syngeneic allograft tumor model was developed using the murine neuroblastoma cell line 9464D derived a tumor from TH-MYCN transgenic mouse. Tumors were generated by transplanting 1 mm3 portions of 9464D flank tumors into the left kidney of C57Bl/6 mice. We investigated the effect of combining HDRT with anti-PD1 antibody on tumor growth and tumor microenvironment. HDRT (8 Gy x 3) was delivered by the small animal radiation research platform (SARRP). Tumor growth was monitored by ultrasound. To assess the effect on immune cells tumors sections were co-imuunostained for six biomarkers using the Vectra multispectral imaging platform. Results: Tumor growth was uniform and confined to the kidney in 100% of transplanted tumors. HDRT was largely restricted to the tumor region with minimal scattered out-of-field dose. Combinatorial treatment with HDRT and PD-1 blockade significantly inhibited tumor growth and prolonged mice survival. We observed augmented T-lymphocyte infiltration, especially CD3+CD8+ lymphocytes, in tumors of mice which received combination treatment. Conclusion: We have developed a novel syngeneic mouse model of MYCN amplified high-risk neuroblastoma. We have utilized this model to show that combining immunotherapy with HDRT inhibits tumor growth and prolongs mice survival.

High-Dose Radiation Increases Notch1 in Tumor Vasculature.

PURPOSE: The aim of this study is to characterize the effects of high-dose radiation therapy (HDRT) on Notch signaling components of the tumor vasculature. METHODS AND MATERIALS: Human umbilical vein endothelial cells monolayers were exposed to different single fraction doses of irradiation; ribonucleic acid RNA was isolated and polymerase chain reaction was performed for Notch signaling components. The vascular response to radiation therapy was examined in a xenograft model of neuroblastoma. Tumors were treated with 0 Gy, 2 Gy, and 12 Gy single fraction doses and analyzed by double immunofluorescence staining for Notch1, Notch ligands Jagged1 and Dll4, and the endothelial cell (EC) marker endomucin. To assess the role of Notch in vivo, NGP xenograft tumors expressing Fc or Notch1-1-24-decoy (a novel Notch inhibitor) were treated with 0 Gy and 12 Gy. Immunofluorescence staining for endomucin and endomucin/αSMA was performed to analyze the effect of combination treatment on tumor EC and endothelial-to-mesenchymal-transition (EndMT), respectively. RESULTS: In human umbilical vein endothelial cells monolayers doses ≥8 Gy increased expression of NOTCH1, JAG1, and Notch target genes HEY1 and HEY2 as early as 6 hours after irradiation. In vivo, 12 Gy significantly increased Notch1 and Jagged1 in tumor ECs compared with 0 Gy or 2 Gy after 72 hours. Combining HDRT with Notch inhibition using the Notch1-1-24-decoy resulted in a greater loss of EC coverage of tumor vessels than HDRT alone at 6 hours and 72 hours post treatment. Notch inhibition reduced EndMT induced by HDRT, as indicated by diminished αSMA staining in ECs. CONCLUSIONS: HDRT induced Notch1 expression and increased Notch1 signaling in the endothelial component of tumor vasculature, which was not observed with lower doses. This increase in Notch1 activation might protect tumor vessels from HDRT induced damage and regulate EndMT process.

Prevalence of Seizures in Pediatric Extracorporeal Membrane Oxygenation Patients as Measured by Continuous Electroencephalography.

OBJECTIVES: Standards for neuromonitoring during extracorporeal membrane oxygenation support do not currently exist, and there is wide variability in practice. We present our institutional experience at an academic children's hospital since establishment of a continuous electroencephalography monitoring protocol for extracorporeal membrane oxygenation patients. DESIGN: Retrospective, single-center study. SETTING: Neonatal ICU and PICU in an urban, quaternary care center. PATIENTS: All neonatal and pediatric patients requiring extracorporeal membrane oxygenation. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: During the study period, 70 patients were cannulated for extracorporeal membrane oxygenation and had continuous electroencephalography monitoring for greater than 24 hours. Electroencephalographic seizures were observed in 16 of 70 patients (23%), including five patients (7%) who were in status epilepticus. Among patients with continuous electroencephalography seizures, nine (56%) had subclinical nonconvulsive status epilepticus and eight (50%) had seizures in the initial 24 hours of extracorporeal membrane oxygenation support. Survival to hospital discharge was significantly greater for extracorporeal membrane oxygenation patients without seizures (74% vs 44%; p = 0.02). CONCLUSIONS: Seizures occur in a significant proportion of pediatric and neonatal extracorporeal membrane oxygenation patients, frequently in the initial 24 hours after extracorporeal membrane oxygenation cannulation. Because seizures are associated with significantly decreased survival, neuromonitoring early in the extracorporeal membrane oxygenation course is important and useful. Further studies are needed to correlate electroencephalography findings with neurologic outcome.

An Analysis of Risk Factors for Hemolysis in Children on Extracorporeal Membrane Oxygenation.

OBJECTIVES: Hemolysis is a known complication of pediatric extracorporeal membrane oxygenation associated with renal failure and mortality. We sought to identify predictors of hemolysis in pediatric extracorporeal membrane oxygenation patients and determine its influence on outcomes. DESIGN: Retrospective, single-center study. SETTING: Urban, quaternary care center pediatric and neonatal ICU. PATIENTS: Ninety-six patients requiring extracorporeal membrane oxygenation. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Daily measurements of plasma-free hemoglobin were obtained while patients were on extracorporeal membrane oxygenation. Patients with a prior extracorporeal membrane oxygenation run, on extracorporeal membrane oxygenation for less than 24 hours, or without complete medical records were excluded from the study. Ninety-six patients met inclusion criteria, of which, 25 patients (26%) had plasma-free hemoglobin greater than 30 mg/dL. Of those patients, 15 of 25(60%) had plasma-free hemoglobin greater than 50 mg/dL, and 21 of 25(84%) occurred during the first 7 days on extracorporeal membrane oxygenation. Compared with patients without hemolysis, those with hemolysis were younger (0.2 mo [0.06-3.2 mo] vs 8.2 mo [0.6-86 mo]; p < 0.001), had a higher pericannulation international normalized ratio (3.9 [3.5-5.5] vs 2.6 [1.8-3.7]; p = 0.003), lower pericannulation platelet count (33 × 10/µL [22-42 × 10/µL] vs 61 × 10/µL [38-86 × 10/µL]; p < 0.001), and had a less negative inlet pressure (-3.5 mm Hg [-14 to 11.5 mm Hg] vs -19 mm Hg [-47 to 0 mm Hg]; p = 0.01). A greater proportion of patients with hemolysis had a heparin assay less than 0.2 mg/dL (50% vs 17%; p = 0.001) and had fluid removal via slow continuous ultrafiltration (32% vs 6%; p < 0.001). Patients with hemolysis had increased risk of in-hospital mortality (odds ratio 10.0; 95% CI 3.4-32; p < 0.001). On multivariable analysis, continuous ultrafiltration (odds ratio, 8.0; 95% CI, 1.9-42; p = 0.007) and pericannulation international normalized ratio greater than 3.5 (odds ratio, 7.2; 95% CI, 2.3-26; p = 0.001) were significantly associated with hemolysis. CONCLUSIONS: Hemolysis is a common complication of pediatric extracorporeal membrane oxygenation. We found that patients with hemolysis (plasma-free hemoglobin > 30 mg/dL) had a 10-fold increase in in-hospital mortality. In our study cohort, hemolysis was associated with continuous ultrafiltration use, but not continuous renal replacement therapy. Additionally, our results suggest that the degree of coagulopathy (international normalized ratio > 3.5) at the time of cannulation influences hemolysis. Additional prospective studies are necessary to define further strategies to prevent hemolysis and improve outcomes in pediatric extracorporeal membrane oxygenation patients.

Demographic and clinical predictors of multiple intracranial aneurysms in patients with subarachnoid hemorrhage.

OBJECTIVE Pathophysiological differences that underlie the development and subsequent growth of multiple aneurysms may exist. In this study, the authors assessed the factors associated with the occurrence of multiple aneurysms in patients presenting with aneurysmal subarachnoid hemorrhage (SAH). METHODS Consecutive patients presenting with aneurysmal SAH between 1996 and 2012 were prospectively enrolled in the Subarachnoid Hemorrhage Outcome Project. Patients harboring 1, 2, or 3 or more aneurysms were stratified into groups, and the clinical and radiological characteristics of each group were compared using multivariate logistic regression. RESULTS Of 1277 patients with ruptured intracranial aneurysms, 890 had 1 aneurysm, 267 had 2 aneurysms, and 120 had 3 or more aneurysms. On multinomial regression using the single-aneurysm cohort as base case, risk factors for patients presenting with 2 aneurysms were female sex (relative risk ratio [RRR] 1.80, p < 0.001), higher body mass index (BMI) (RRR 1.02, p = 0.003), more years of smoking (RRR = 1.01, p = 0.004), and black race (RRR 1.83, p = 0.001). The risk factors for patients presenting with 3 or more aneurysms were female sex (RRR 3.10, p < 0.001), higher BMI (RRR 1.03, p < 0.001), aneurysm in the posterior circulation (RRR 2.59, p < 0.001), and black race (RRR 2.15, p = 0.001). Female sex, longer smoking history, aneurysms in the posterior circulation, BMI, and black race were independently associated with the development of multiple aneurysms in our adjusted multivariate multinomial model. CONCLUSIONS Significant demographic and clinical differences are found between patients presenting with single and multiple aneurysms in the setting of aneurysmal SAH. These predictors of multiple aneurysms likely reflect a predisposition toward inflammation and endothelial injury.

High-Dose, Single-Fraction Irradiation Rapidly Reduces Tumor Vasculature and Perfusion in a Xenograft Model of Neuroblastoma.

PURPOSE: To characterize the effects of high-dose radiation therapy (HDRT) on neuroblastoma tumor vasculature, including the endothelial cell (EC)-pericyte interaction as a potential target for combined treatment with antiangiogenic agents. METHODS AND MATERIALS: The vascular effects of radiation therapy were examined in a xenograft model of high-risk neuroblastoma. In vivo 3-dimensional contrast-enhanced ultrasonography (3D-CEUS) imaging and immunohistochemistry (IHC) were performed. RESULTS: HDRT significantly reduced tumor blood volume 6 hours after irradiation compared with the lower doses used in conventionally fractionated radiation. There was a 63% decrease in tumor blood volume after 12-Gy radiation compared with a 24% decrease after 2 Gy. Analysis of tumor vasculature by lectin angiography showed a significant loss of small vessel ends at 6 hours. IHC revealed a significant loss of ECs at 6 and 72 hours after HDRT, with an accompanying loss of immature and mature pericytes at 72 hours. CONCLUSIONS: HDRT affects tumor vasculature in a manner not observed at lower doses. The main observation was an early reduction in tumor perfusion resulting from a reduction of small vessel ends with a corresponding loss of endothelial cells and pericytes.

Transnasal endoscopic approach to the pediatric craniovertebral junction and rostral cervical spine: case series and literature review.

The endoscopic transnasal approach to the rostral pediatric spine and craniovertebral junction is a relatively new technique that provides an alternative to the traditional transoral approach to the anterior pediatric spine. In this case series, the authors provide 2 additional examples of patients undergoing endoscopic transnasal odontoidectomies for ventral decompression of the spinal cord. Both patients would have required transection of the palate to undergo an effective transoral operation, which can be a cause of significant morbidity. In one case, transnasal decompression was initially incomplete, and decompression was successfully achieved via a second endoscopic transnasal operation. Both cases resulted in significant neurological recovery and stable long-term spinal alignment. The transnasal approach benefits from entering into the posterior pharynx at an angle that often reduces the length of postoperative intubation and may speed a patient's return to oral intake. Higher reoperation rates are a concern for many endoscopic approaches, but there are insufficient data to conclude if this is the case for this procedure. Further experience with this technique will provide a better understanding of the indications for which it is most effective. Transcervical and transoral endoscopic approaches have also been reported and provide additional options for pediatric anterior cervical spine surgery.

The role of advanced neuroimaging in intracerebral hemorrhage.

In this report, the authors sought to summarize existing literature to provide an overview of the currently available techniques and to critically assess the evidence for or against their application in intracerebral hemorrhage (ICH) for management, prognostication, and research. Functional imaging in ICH represents a potential major step forward in the ability of physicians to assess patients suffering from this devastating illness due to the advantages over standing imaging modalities focused on general tissue structure alone, but its use is highly controversial due to the relative paucity of literature and the lack of consolidation of the predominantly small data sets that are currently in existence. Current data support that diffusion tensor imaging and tractography, diffusion-perfusion weighted MRI techniques, and functional MRI all possess major potential in the areas of highlighting motor deficits, motor recovery, and network reorganization. Novel clinical studies designed to objectively assess the value of each of these modalities on a wider scale in conjunction with other methods of investigation and management will allow for their rapid incorporation into standard practice.

The length dependence of the polyQ-mediated protein aggregation.

Polyglutamine (polyQ) repeat disorders are caused by the expansion of CAG tracts in certain genes, resulting in transcription of proteins with abnormally long polyQ inserts. When these inserts expand beyond 35-45 glutamines, affected proteins form toxic aggregates, leading to neuron death. Chymotrypsin inhibitor 2 (CI2) with an inserted glutamine repeat has previously been used to model polyQ-mediated aggregation in vitro. However, polyQ insertion lengths in these studies have been kept below the pathogenic threshold. We perform molecular dynamics simulations to study monomer folding dynamics and dimer formation in CI2-polyQ chimeras with insertion lengths of up to 80 glutamines. Our model recapitulates the experimental results of previous studies of chimeric CI2 proteins, showing high folding cooperativity of monomers as well as protein association via domain swapping. Surprisingly, for chimeras with insertion lengths above the pathogenic threshold, monomer folding cooperativity decreases and the dominant mode for dimer formation becomes interglutamine hydrogen bonding. These results support a mechanism for pathogenic polyQ-mediated aggregation, in which expanded polyQ tracts destabilize affected proteins and promote the formation of partially unfolded intermediates. These unfolded intermediates form aggregates through associations by interglutamine interactions.