Survival outcomes in pediatric patients with metastatic Ewing sarcoma who achieve a rapid complete response of pulmonary metastases.

PURPOSE: Our objectives were to compare overall survival (OS) and pulmonary relapse between patients with metastatic Ewing sarcoma (EWS) at diagnosis who achieve rapid complete response (RCR) and those with residual pulmonary nodules after induction chemotherapy (non-RCR). PATIENTS AND METHODS: This retrospective cohort study included children under 20 years with metastatic EWS treated from 2007 to 2020 at 19 institutions in the Pediatric Surgical Oncology Research Collaborative. Chi-square tests were conducted for differences among groups. Kaplan-Meier curves were generated for OS and pulmonary relapse. RESULTS: Among 148 patients with metastatic EWS at diagnosis, 61 (41.2%) achieved RCR. Five-year OS was 71.2% for patients who achieved RCR, and 50.2% for those without RCR (p = .04), and in multivariable regression among patients with isolated pulmonary metastases, RCR (hazards ratio [HR] 0.42; 95% confidence interval [CI]: 0.17-0.99) and whole lung irradiation (WLI) (HR 0.35; 95% CI: 0.16-0.77) were associated with improved survival. Pulmonary relapse occurred in 57 (37%) patients, including 18 (29%) in the RCR and 36 (41%) in the non-RCR groups (p = .14). Five-year pulmonary relapse rates did not significantly differ based on RCR (33.0%) versus non-RCR (47.0%, p = .13), or WLI (38.8%) versus no WLI (46.0%, p = .32). DISCUSSION: Patients with EWS who had isolated pulmonary metastases at diagnosis had improved OS if they achieved RCR and received WLI, despite having no significant differences in rates of pulmonary relapse.

Physiology of chemokines in the cancer microenvironment.

Chemokines are chemotactic cytokines whose canonical functions govern movement of receptor-expressing cells along chemical gradients. Chemokines are a physiological system that is finely tuned by ligand and receptor expression, ligand or receptor oligomerization, redundancy, expression of atypical receptors, and non-GPCR binding partners that cumulatively influence discrete pharmacological signaling responses and cellular functions. In cancer, chemokines play paradoxical roles in both the directed emigration of metastatic, receptor-expressing cancer cells out of the tumor as well as immigration of tumor-infiltrating immune cells that culminate in a tumor-unique immune microenvironment. In the age of precision oncology, strategies to effectively harness the power of immunotherapy requires consideration of chemokine gradients within the unique spatial topography and temporal influences with heterogeneous tumors. In this article, we review current literature on the diversity of chemokine ligands and their cellular receptors that detect and process chemotactic gradients and illustrate how differences between ligand recognition and receptor activation influence the signaling machinery that drives cellular movement into and out of the tumor microenvironment. Facets of chemokine physiology across discrete cancer immune phenotypes are contrasted to existing chemokine-centered therapies in cancer.

Multi-institutional assessment of the prevalence of neuroendocrine tumors in children undergoing laparoscopic appendectomy for acute appendicitis in the United States.

As non-operative management of acute appendicitis in children has become more common, missed incidental appendiceal pathology can be an unintended consequence. We assessed the prevalence of neuroendocrine tumors in appendectomy specimens from eight US children's hospitals from 2012 to 2021. The prevalence of neuroendocrine tumors (NET) was found to be 1:271, with a median age of 14 years and 62% female. Most tumors were small (median 6 mm; interquartile range [IQR]: 3-10), and no recurrence was noted during the follow-up period (median 22.5 months; IQR: 3-53). The possibility of delayed diagnosis of these tumors should be part of the discussion for non-operative management of pediatric acute appendicitis.

Impact of Referring Hospital Imaging on Mortality at a Level I Trauma Center.

BACKGROUND: Computed tomography (CT) has become a standard adjunct in the evaluation of patients with trauma. However, utility of imaging at the referring hospital remains controversial. We study the effect of CT scans at referring hospitals on in-hospital mortality at a receiving trauma center. MATERIALS AND METHODS: A retrospective cohort study was performed with adult patients with severe trauma transferred to a level I trauma center from regional nontrauma hospitals between 2012 and 2017. Baseline characteristics were compared with Student's t-test and Pearson's chi-squared testing. The primary endpoint was in-hospital mortality. Cox regression, controlling for transfer time, was used to evaluate the effect of imaging on mortality. RESULTS: Three thousand four hundred and fifteen adult patients with trauma were included: 1135 (33.2%) received a pretransfer CT scan, whereas 2280 (66.8%) did not. Patients who received a pretransfer CT scan were more likely to be older, female, white, have a higher Charlson Comorbidity Index, less severely injured, have a blunt mechanism, and be transferred by ground. There was no difference in distance (58.3 miles versus 57.0 miles, P = 0.34), but transfer times were significantly increased for those who received pretransfer scans (288 versus 213 min, P < 0.005). The adjusted model controlling for multiple variables has a hazard ratio of 0.533 (95% confidence interval 0.42-0.68, P < 0.005). CONCLUSIONS: There is a survival advantage for patients who receive pretransfer CT scans despite having significantly longer transport times. We suggest that this decreased mortality associated with pretransfer imaging may reflect improving trends in referring physician transfer decisions.

Induced differentiation inhibits sphere formation in neuroblastoma.

Neuroblastoma arises from the neural crest, the precursor cells of the sympathoadrenal axis, and differentiation status is a key prognostic factor used for clinical risk group stratification and treatment strategies. Neuroblastoma tumor-initiating cells have been successfully isolated from patient tumor samples and bone marrow using sphere culture, which is well established to promote growth of neural crest stem cells. However, accurate quantification of sphere-forming frequency of commonly used neuroblastoma cell lines has not been reported. Here, we show that MYCN-amplified neuroblastoma cell lines form spheres more frequently than non-MYCN-amplified cell lines. We also show that sphere formation is directly sensitive to cellular differentiation status. 13-cis-retinoic acid is a clinically used differentiating agent that induces a neuronal phenotype in neuroblastoma cells. Induced differentiation nearly completely blocked sphere formation. Furthermore, sphere formation was specifically FGF-responsive and did not respond to increasing doses of EGF. Taken together, these data suggest that sphere formation is an accurate method of quantifying the stemness phenotype in neuroblastoma.

A critical role for TLR4 induction of autophagy in the regulation of enterocyte migration and the pathogenesis of necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) develops in response to elevated TLR4 signaling in the newborn intestinal epithelium and is characterized by TLR4-mediated inhibition of enterocyte migration and reduced mucosal healing. The downstream processes by which TLR4 impairs mucosal healing remain incompletely understood. In other systems, TLR4 induces autophagy, an adaptive response to cellular stress. We now hypothesize that TLR4 induces autophagy in enterocytes and that TLR4-induced autophagy plays a critical role in NEC development. Using mice selectively lacking TLR4 in enterocytes (TLR4(ΔIEC)) and in TLR4-deficient cultured enterocytes, we now show that TLR4 activation induces autophagy in enterocytes. Immature mouse and human intestine showed increased expression of autophagy genes compared with full-term controls, and NEC development in both mouse and human was associated with increased enterocyte autophagy. Importantly, using mice in which we selectively deleted the autophagy gene ATG7 from the intestinal epithelium (ATG7(ΔIEC)), the induction of autophagy was determined to be required for and not merely a consequence of NEC, because ATG7(ΔIEC) mice were protected from NEC development. In defining the mechanisms involved, TLR4-induced autophagy led to impaired enterocyte migration both in vitro and in vivo, which in cultured enterocytes required the induction of RhoA-mediated stress fibers. These findings depart from current dogma in the field by identifying a unique effect of TLR4-induced autophagy within the intestinal epithelium in the pathogenesis of NEC and identify that the negative consequences of autophagy on enterocyte migration play an essential role in its development.

Targeted immune activation in pediatric solid tumors: opportunities to complement local control approaches.

Surgery or radiation therapy is nearly universally applied for pediatric solid tumors. In many cases, in diverse tumor types, distant metastatic disease is present and evades surgery or radiation. The systemic host response to these local control modalities may lead to a suppression of antitumor immunity, with potential negative impact on the clinical outcomes for patients in this scenario. Emerging evidence suggests that the perioperative immune responses to surgery or radiation can be modulated therapeutically to preserve anti-tumor immunity, with the added benefit of preventing these local control approaches from serving as pro-tumorigenic stimuli. To realize the potential benefit of therapeutic modulation of the systemic response to surgery or radiation on distant disease that evades these modalities, a detailed knowledge of the tumor-specific immunology as well as the immune responses to surgery and radiation is imperative. In this Review we highlight the current understanding of the tumor immune microenvironment for the most common peripheral pediatric solid tumors, the immune responses to surgery and radiation, and current evidence that supports the potential use of immune activating agents in the perioperative window. Finally, we define existing knowledge gaps that limit the current translational potential of modulating perioperative immunity to achieve effective anti-tumor outcomes.

Isoxazole compound ML327 blocks MYC expression and tumor formation in neuroblastoma.

Neuroblastomas are the most common extracranial solid tumors in children and arise from the embryonic neural crest. MYCN-amplification is a feature of ∼30% of neuroblastoma tumors and portends a poor prognosis. Neural crest precursors undergo epithelial-to-mesenchymal transition (EMT) to gain migratory potential and populate the sympathoadrenal axis. Neuroblastomas are posited to arise due to a blockade of neural crest differentiation. We have recently reported effects of a novel MET inducing compound ML327 (N-(3-(2-hydroxynicotinamido) propyl)-5-phenylisoxazole-3-carboxamide) in colon cancer cells. Herein, we hypothesized that forced epithelial differentiation using ML327 would promote neuroblastoma differentiation. In this study, we demonstrate that ML327 in neuroblastoma cells induces a gene signature consistent with both epithelial and neuronal differentiation features with adaptation of an elongated phenotype. These features accompany induction of cell death and G1 cell cycle arrest with blockage of anchorage-independent growth and neurosphere formation. Furthermore, pretreatment with ML327 results in persistent defects in proliferative potential and tumor-initiating capacity, validating the pro-differentiating effects of our compound. Intriguingly, we have identified destabilization of MYC signaling as an early and consistent feature of ML327 treatment that is observed in both MYCN-amplified and MYCN-single copy neuroblastoma cell lines. Moreover, ML327 blocked MYCN mRNA levels and tumor progression in established MYCN-amplified xenografts. As such, ML327 may have potential efficacy, alone or in conjunction with existing therapeutic strategies against neuroblastoma. Future identification of the specific intracellular target of ML327 may inform future drug discovery efforts and enhance our understanding of MYC regulation.

FX11 inhibits aerobic glycolysis and growth of neuroblastoma cells.

BACKGROUND: The MYC family of proteins promotes neuroblastoma tumorigenesis at least in part through the induction of aerobic glycolysis by promoting the transcription of key glycolytic enzymes, such as LDHA. FX11 is a selective inhibitor of LDHA that has demonstrated preclinical efficacy in adult cancers. Herein, we hypothesized that FX11 would inhibit aerobic glycolysis and block growth of neuroblastoma cells. METHODS: We surveyed 3 MYCN-single copy and 5 MYCN-amplified neuroblastoma cell lines to correlate C-MYC/N-MYC protein levels with LDHA expression. Cell viability was measured with FX11 using a tetrazolium-based assay. Cell cycle analysis using propidium iodide with flow cytometry was performed to evaluate for growth arrest. Immunoblotting demonstrated PARP and Caspase 3 cleavage as evidence of apoptosis. RESULTS: LDHA is frequently expressed in both MYCN--amplified and MYCN-single copy cell lines. N-MYC and C-MYC protein levels did not correlate with LDHA protein expression. FX11 inhibits aerobic glycolysis and growth in three MYCN-amplified and one MYCN-single copy neuroblastoma cell lines. FX11 induces modest G1 cell cycle arrest with selective induction of apoptosis. CONCLUSION: Small molecule LDHA inhibition is capable of blocking aerobic glycolysis and growth of neuroblastoma cell lines in vitro and merits further in vivo evaluation of its preclinical efficacy in neuroblastomas.

Laparoscopic Nissen fundoplication in infants with hypoplastic left heart syndrome.

BACKGROUND/PURPOSE: Patients with hypoplastic left heart syndrome (HLHS) experience a higher risk for complications from gastroesophageal reflux, prompting frequent need for fundoplication. Patients between stage I and II palliation ("interstage") are at particularly high operative risk because of the parallel nature of their pulmonary and systemic blood flow. Laparoscopic approach for fundoplication is common for pediatric patients. However, its safety in interstage HLHS is relatively unknown. We examined the perioperative physiologic burden of a laparoscopic fundoplication in HLHS patients. METHODS: All patients who underwent open or laparoscopic fundoplication during the interstage period at our institution since 2006 were reviewed. Perioperative physiologic data, echocardiographic findings, survival, and complications were collected from the anesthetic record and patient chart. RESULTS: Nineteen patients with HLHS had laparoscopic fundoplication, 13 (68%) during the interstage period, compared to 64 performed by the open approach. Ten (77%) of 13 interstage patients had perioperative hemodynamic instability. Incidence of instability between open and laparoscopic groups was not different. One laparoscopic patient required ECMO support for shunt thrombosis. CONCLUSIONS: Despite a high incidence of hemodynamic instability, overall outcomes are consistent with those reported in the literature for this high-risk patient population. Laparoscopic approach for fundoplication during the interstage period appears to be a relatively safe option for these patients.

Antioxidant inhibition of steady-state reactive oxygen species and cell growth in neuroblastoma.

BACKGROUND: Reactive oxygen species (ROS) contribute to adult tumorigenesis; however, their roles in pediatric solid tumors are unknown. Here, we sought to define the steady-state ROS levels in neuroblastoma and to examine whether aggressive cellular behavior, which may predict treatment failure, is regulated by ROS. METHODS: Neuroblastoma sections were assessed for 4-hydroxynonenal (4-HNE), a marker of intracellular lipid peroxidation and a byproduct of increased levels of ROS. Human neuroblastoma cell lines, MYCN-amplified BE(2)-C and MYCN-nonamplified SK-N-SH, were examined in our study. Superoxide and hydroperoxide oxidation products were detected by staining for dihydroethidium (DHE) and 5, 6-carboxy-2', 7'-dichlorodihydrofluorescein diacetate (CDCFH2), using the oxidation-insensitive analog CDCF as a negative control. Cells were treated with N-acetylcysteine (NAC; 10 mmol/L) daily for 5 days and analyzed. RESULTS: Greater expression of 4-HNE was observed in undifferentiated tumor sections as compared with the more differentiated tumors. Interestingly, increased levels of ROS were detected in MYCN-amplified BE(2)-C cells. Moreover, gastrin-releasing peptide receptor-induced ROS production stimulated upregulation of the hypoxia inducible factor (HIF)-1α/vascular endothelial growth factor (VEGF) pathway and an increase in cell growth. Antioxidant NAC decreased HIF-1α/VEGF expression and inhibited BE(2)-C cell growth. CONCLUSION: We report a novel observation that shifting the redox balance toward greater ROS levels results in a more aggressive neuroblastoma phenotype. Our data suggest that ROS play a critical role in refractory neuroblastoma.

Bromodomain and extraterminal inhibition blocks tumor progression and promotes differentiation in neuroblastoma.

BACKGROUND: MYCN amplification is a key molecular hallmark of high-risk neuroblastoma. Previously considered an "undruggable" target, MYCN transcription can be disrupted by inhibiting the bromodomain and the extraterminal (BET) domain family of proteins that regulates MYCN transcription epigenetically. JQ1 is a potent, small-molecule BET inhibitor that induces cell-cycle arrest and initiates apoptosis in neuroblastoma. Here, we sought to validate the antitumorigenic effects of JQ1 in neuroblastoma and to evaluate whether blocking N-myc expression with JQ1 promotes neural differentiation. METHODS: We determined the effects in vitro of JQ1 treatment on human neuroblastoma cell growth in both monolayer and sphere-forming conditions. Subcutaneous neuroblastoma xenografts were used for an in vivo study. Western blotting and immunohistochemistry were performed to evaluate the effects on neural differentiation and stem cell markers. RESULTS: JQ1 treatment blocked neuroblastoma cell growth in both monolayer and sphere-forming conditions; JQ1 also attenuated the growth of neuroblastoma xenograft in athymic nude mice. Neurofilament expression was enhanced with JQ1 treatment, indicating that JQ1 induces neuronal differentiation. Sphere forming conditions resulted in increased expression of multiple stem cell markers; these effects were reversed with JQ1 treatment. CONCLUSION: BET inhibition attenuates progression and promotes neural differentiation of neuroblastoma in vitro and in vivo in mice, providing insight into potential clinical applications of BET inhibitors in the treatment of patients with neuroblastoma.

Silencing of CDC42 inhibits neuroblastoma cell proliferation and transformation.

Cell division cycle 42 (CDC42), a small GTPase of the Rho-subfamily, regulates diverse cellular functions including proliferation, cytoskeletal rearrangement and even promotes malignant transformation. Here, we found that increased expression of CDC42 correlated with undifferentiated neuroblastoma as compared to a more benign phenotype. CDC42 inhibition decreased cell growth and soft agar colony formation, and increased cell death in BE(2)-C and BE(2)-M17 cell lines, but not in SK-N-AS. In addition, silencing of CDC42 decreased expression of N-myc in BE(2)-C and BE(2)-M17 cells. Our findings suggest that CDC42 may play a role in the regulation of aggressive neuroblastoma behavior.

Endorectal pull-through for Hirschsprung's disease-a multicenter, long-term comparison of results: transanal vs transabdominal approach.

PURPOSE: Previous studies have reported decreased continence in patients undergoing transanal endorectal pull-through (TERP) for Hirschsprung's disease compared to the older transabdominal approach (TAA). To address this, we examined long-term stooling outcomes in a large, multicenter cohort of patients undergoing either TERP or TAA. METHODS: Data were collected from 5 large pediatric institutions. Patient families were surveyed using a stooling score system (0-40, best to worst total score). Inclusion criteria included patients older than 3 years and those who had more than 6 months of recovery after pull-through. Those with total colonic aganglionosis were excluded. Statistical analysis included univariate and multivariate linear regression (significance, P < .05). RESULTS: Two hundred eighty-one patients underwent TERP (192) or TAA (89). Interviews were completed in 149 (104 [52%] TERP vs 45 [52%] TAA). The TAA group had a significantly greater number of daily bowel movements for each respective postoperative year and experienced more early complications (3% vs 1% with >1 complication; P = .061) and late complications (19% vs 4% with >1 complication; P < .001). Although the TAA group had a higher mean enterocolitis score (3.3 +/- 0.4 vs 1.8 +/- 0.2; P < .001), this was not borne out by multivariate regression analysis (P = .276). Parental survey showed that there were no significant differences between procedures in mean total, continence, or stooling pattern scores. CONCLUSION: Transanal endorectal pull-through was associated with fewer complications and fewer episodes of enterocolitis. In contrast to prior studies, TERP patients did not have a higher rate of incontinence. These results support use of TERP as an excellent surgical approach for children with Hirschsprung's disease.