Activating Transcription Factor 5 Promotes Neuroblastoma Metastasis by Inducing Anoikis Resistance.

MYCN-amplified neuroblastoma often presents as a highly aggressive metastatic disease with a poor prognosis. Activating transcription factor 5 (ATF5) is implicated in neural cell differentiation and cancer cell survival. Here, we show that ATF5 is highly expressed in patients with stage 4 high-risk neuroblastoma, with increased expression correlating with a poorer prognosis. We demonstrated that ATF5 promotes the metastasis of neuroblastoma cell lines in vivo. Functionally, ATF5 depletion significantly reduced xenograft tumor growth and metastasis of neuroblastoma cells to the bone marrow and liver. Mechanistically, ATF5 endows tumor cells with resistance to anoikis, thereby increasing their survival in systemic circulation and facilitating metastasis. We identified the proapoptotic BCL-2 modifying factor (BMF) as a critical player in ATF5-regulated neuroblastoma anoikis. ATF5 suppresses BMF under suspension conditions at the transcriptional level, promoting anoikis resistance, whereas BMF knockdown significantly prevents ATF5 depletion-induced anoikis. Therapeutically, we showed that a cell-penetrating dominant-negative ATF5 peptide, CP-d/n-ATF5, inhibits neuroblastoma metastasis to the bone marrow and liver by inducing anoikis sensitivity in circulating tumor cells. Our study identified ATF5 as a metastasis promoter and CP-d/n-ATF5 as a potential antimetastatic therapeutic agent for neuroblastoma. SIGNIFICANCE: This study shows that resistance to anoikis in neuroblastoma is mediated by ATF5 and offers a rationale for targeting ATF5 to treat metastatic neuroblastoma.

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.

Characterization of Extracorporeal Membrane Oxygenation Support for Single Ventricle Patients.

BACKGROUND: Extracorporeal membrane oxygenation (ECMO) can provide crucial support for single ventricle (SV) patients at various stages of palliation. However, characterization of the utilization and outcomes of ECMO in these unique patients remains incompletely studied. METHODS: We performed a single-center retrospective review of SV patients between 2010 and 2017 who underwent ECMO cannulation with primary end point of survival to discharge and secondary end point of survival to decannulation or orthotopic heart transplantation (OHT). Multivariate analysis was performed for factors predictive of survival to discharge and survival to decannulation. RESULTS: Forty SV patients with a median age of one month (range: 3 days to 15 years) received ECMO support. The incidence of ECMO was 14% for stage I, 3% for stage II, and 4% for stage III. Twenty-seven (68%) patients survived to decannulation, and 21 (53%) patients survived to discharge, with seven survivors to discharge undergoing OHT. Complications included infection (40%), bleeding (40%), thrombosis (33%), and radiographic stroke (45%). Factors associated with survival to decannulation included pre-ECMO lactate (hazard ratio [HR]: 0.61, 95% confidence interval [CI]: 0.41-0.90, P = .013) and post-ECMO bicarbonate (HR: 1.24, 95% CI: 1.0-1.5, P = .018). Factors associated with survival to discharge included central cannulation (HR: 40.0, 95% CI: 3.1-500.0, P = .005) and lack of thrombotic complications (HR: 28.7, 95% CI: 2.1-382.9, P = .011). CONCLUSIONS: Extracorporeal membrane oxygenation can be useful to rescue SV patients with approximately half surviving to discharge, although complications are frequent. Early recognition of the role of heart transplant is imperative. Further study is required to identify areas for improvement in this population.

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.

Extracorporeal Cardiopulmonary Resuscitation (ECPR) in Infants and Children: A Single-Center Retrospective Study.

BACKGROUND: Extracorporeal cardiopulmonary resuscitation (ECPR) is increasingly used to rescue patients with cardiac arrest refractory to conventional therapy, necessitating evaluation of factors that may affect outcomes. METHODS: A single-center retrospective review of pediatric patients (<21 years old) who underwent ECPR from January 2010 to November 2017. Comparisons between nonsurvivors and survivors, to decannulation and discharge, were made. Factors associated with survival and rate of complications were examined. RESULTS: Seventy patients were supported with ECPR. Forty-nine (70%) patients survived to decannulation and 38 (54%) patients to discharge. There was no statistical difference between baseline characteristics of survivors and nonsurvivors, including age at cannulation, weight (kg), time to cannulation (minutes), and total time on extracorporeal membrane oxygenation (hours). Survivors to discharge had significantly higher pH prior to cannulation compared to nonsurvivors (7.11 ± 0.24 vs 6.97 ± 0.21, P = .01). Of all, 23.2% of patients received renal replacement therapy (RRT), 39.4% had significant bleeding, 22.5% had thrombotic complications, and 68.8% had neurologic injury on imaging studies. A greater number of nonsurvivors received RRT compared to survivors to discharge (35.5% vs 10.8%, P = .02). There were no differences in bleeding or thrombotic complications or radiographically established neurologic injury. CONCLUSIONS: Although ECPR effectively increases overall survival, a better characterization of long-term outcomes is needed.

Pressure ulcers in paediatric patients on extracorporeal membrane oxygenation.

It has been shown that pressure ulcer formation in critically ill paediatric patients increases morbidity and mortality. We sought to identify factors associated with pressure ulcer formation in paediatric patients on extracorporeal membrane oxygenation (ECMO). From December 2014 to 2015, we identified patients at our institution who developed a pressure ulcer to create two cohorts: ulcer and no ulcer. Variables of interest included: type of ECMO, ECMO indication, hours on ECMO, location of cannulas, volume of crystalloid and blood products received during the first 7 days or during the length of the ECMO run, albumin and lactate levels on the day of cannulation, and presence of vasopressor support or steroid therapy. Of 43 patients studied, 11 (25.5%) developed a pressure ulcer. Patients that developed ulcers were older (P = 0.001) and weighed more (P = 0.006). Femoral cannulation was more frequent in the ulcer group (36.4% vs 6.3%, P = 0.029), and duration of ECMO was longer (P = 0.007). Age, weight, duration of ECMO, and femoral cannulation may contribute to the development of pressure ulcers in children who require ECMO support. Further analysis is imperative to identify specific techniques and protocols that will prevent pressure ulcers in this critically ill population.

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.

Routine Use of Distal Arterial Perfusion in Pediatric Femoral Venoarterial Extracorporeal Membrane Oxygenation.

Lower-extremity ischemia is a significant complication in children on femoral venoarterial extracorporeal membrane oxygenation (VA ECMO). Our institution currently routinely uses distal perfusion catheters (DPCs) in all femoral arterial cannulations in attempts to reduce ischemia. We performed a single-center, retrospective review of pediatric patients supported with femoral VA ECMO from January 2005 to November 2015. The outcomes of patients with prophylactic DPC placement at cannulation (prophylactic DPC) were compared to a historical group with DPCs placed in response only to clinically evident ischemic changes (reactive DPC). Ischemic complication requiring invasive intervention (fasciotomy or amputation) was the primary outcome. Twenty-nine patients underwent a total of 31 femoral arterial cannulations, 17 with prophylactic DPC and 14 with reactive DPC. Ischemic complications requiring invasive intervention developed in 2 of 17 (12%) prophylactic DPC patients versus 4 of 14 (29%) reactive DPC. In the reactive DPC group, 7 of 14 (50%) had ischemic changes postcannulation, six underwent DPC placement, and three out of six of these patients still required invasive intervention. One of the seven patients had ischemic changes, did not undergo DPC, and required amputation. While a greater percentage of patients in the prophylactic group was cannulated during extracorporeal cardiopulmonary resuscitation (ECPR), statistical significance was not otherwise demonstrated. We demonstrate feasibility of superficial femoral artery (SFA) access in pediatric patients. We note fewer ischemic complications with prophylactic DPC placement, and observe that salvaging a limb with a reactive DPC was only successful 50% of the time. Although there was no statistical difference in the primary outcome between the two groups, limitations and confounding factors include small sample size and a greater percentage of patients in the prophylactic DPC group cannulated with ECPR in progress.

Validation of the Surgical Apgar Score in a veteran population undergoing general surgery.

BACKGROUND: The Surgical Apgar Score (SAS, a 10-point score calculated using limited intraoperative data) can correlate with postoperative morbidity and mortality after general surgery. We evaluated reliability of SAS in a veteran population. STUDY DESIGN: We prospectively collected demographics, medical history, type of surgery, and postoperative outcomes for any veteran undergoing general surgery at our institution (2006-2011). We categorized patients in 4 SAS groups and compared differences in morbidity and mortality. RESULTS: Our study population included 2,125 patients (SAS ≤4: n = 29; SAS 5-6: n = 227; SAS 7-8: n = 797; SAS 9-10: n = 1,072). Low-SAS patients were likely to have significant preoperative comorbidities and to undergo major surgery, and had increased postoperative morbidity and 30-day mortality. CONCLUSIONS: The SAS is easily calculated from 3 routinely available intraoperative measurements, correlates with fixed preoperative risk (acute conditions, pre-existing comorbidities, operative complexity), and effectively identifies veterans at high risk for postoperative complications.