Optimising the chick chorioallantoic membrane xenograft model of neuroblastoma for drug delivery

Background Neuroblastoma is a paediatric cancer that despite multimodal therapy still has a poor outcome for many patients with high risk tumours. Retinoic acid (RA) promotes differentiation of some neuroblastoma tumours and cell lines, and is successfully used clinically, supporting the view that differentiation therapy is a promising strategy for treatment of neuroblastoma. To improve treatment of a wider range of tumour types, development and testing of novel differentiation agents is essential. New pre-clinical models are therefore required to test therapies in a rapid cost effective way in order to identify the most useful agents. Methods As a proof of principle, differentiation upon ATRA treatment of two MYCN-amplified neuroblastoma cell lines, IMR32 and BE2C, was measured both in cell cultures and in tumours formed on the chick chorioallantoic membrane (CAM). Differentiation was assessed by 1) change in cell morphology, 2) reduction in cell proliferation using Ki67 staining and 3) changes in differentiation markers (STMN4 and ROBO2) and stem cell marker (KLF4). Results were compared to MLN8237, a classical Aurora Kinase A inhibitor. For the in vivo experiments, cells were implanted on the CAM at embryonic day 7 (E7), ATRA treatment was between E11 and E13 and tumours were analysed at E14. Results Treatment of IMR32 and BE2C cells in vitro with 10 μM ATRA resulted in a change in cell morphology, a 65% decrease in cell proliferation, upregulation of STMN4 and ROBO2 and downregulation of KLF4. ATRA proved more effective than MLN8237 in these assays. In vivo, 100 μM ATRA repetitive treatment at E11, E12 and E13 promoted a change in expression of differentiation markers and reduced proliferation by 43% (p < 0.05). 40 μM ATRA treatment at E11 and E13 reduced proliferation by 37% (p < 0.05) and also changed cell morphology within the tumour. Conclusion Differentiation of neuroblastoma tumours formed on the chick CAM can be analysed by changes in cell morphology, proliferation and gene expression. The well-described effects of ATRA on neuroblastoma differentiation were recapitulated within 3 days in the chick embryo model, which therefore offers a rapid, cost effective model compliant with the 3Rs to select promising drugs for further preclinical analysis

Crizotinib-induced antitumour activity in human alveolar rhabdomyosarcoma cells is not solely dependent on ALK and MET inhibition

BACKGROUND: Rhabdomyosarcoma (RMS) is the most commonly diagnosed malignant soft tissue tumour in children and adolescents. Aberrant expression of Anaplastic Lymphoma Kinase (ALK) and MET gene has been implicated in the malignant progression of RMS, especially in the alveolar subtype. This observation suggests that crizotinib (PF-02341066), a kinase inhibitor against ALK and MET, may have a therapeutic role in RMS, although its antitumour activity in this malignancy has not yet been studied. METHODS: RH4 and RH30 alveolar RMS (ARMS) cell lines were treated with crizotinib and then assessed by using proliferation, viability, migration and colony formation assays. Multiple approaches, including flow cytometry, immunofluorescence, western blotting and siRNA-based knock-down, were used in order to investigate possible molecular mechanisms linked to crizotinib activity. RESULTS: In vitro treatment with crizotinib inhibited ALK and MET proteins, as well as Insulin-like Growth Factor 1 Receptor (IGF1R), with a concomitant robust dephosphorylation of AKT and ERK, two downstream kinases involved in RMS cell proliferation and survival. Exposure to crizotinib impaired cell growth, and accumulation at G2/M phase was attributed to an altered expression and activation of checkpoint regulators, such as Cyclin B1 and Cdc2. Crizotinib was able to induce apoptosis and autophagy in a dose-dependent manner, as shown by caspase-3 activation/PARP proteolytic cleavage down-regulation and by LC3 activation/p62 down-regulation, respectively. The accumulation of reactive oxygen species (ROS) seemed to contribute to crizotinib effects in RH4 and RH30 cells. Moreover, crizotinib-treated RH4 and RH30 cells exhibited a decreased migratory/invasive capacity and clonogenic potential. CONCLUSIONS: These results provide a further insight into the molecular mechanisms affected by crizotinib in ARMS cells inferring that it could be a useful therapeutic tool in ARMS cancer treatment

Postoperative feeding in neonatal duodenal obstruction

BackgroundFindings from manometry studies and contrast imaging reveal functioning gastric physiology in newborns with duodenal atresia and stenosis. Stomach reservoir function should therefore be valuable in aiding the postoperative phase of gastric feeding. The aim of this study was therefore to compare the feasibility of initiating oral or large volume(s) gavage feeds vs small volume bolus feeds following operation for congenital duodenal anomalies.MethodsSingle-center electronic medical records of all babies with duodenal atresia and stenosis admitted to a university surgical center during January 1997-September 2021 were analyzed. A fast-fed group (FF) included newborns fed with oral or gavage feeds advanced at a rate of at least 2.5 ml/kg and then progressed more than once a day vs slow-fed group (SF) fed with gavage feeds at incremental rate less than 2.5 ml/kg/day for each time period of oral tolerance or by drip feeds. Total feed volume was limited to 120-150 ml/kg/day in the respective study cohort populations.ResultsFifty-one eligible patients were recruited in the study - twenty-six in FF group and twenty-five in SF group. Statistically significant differences were observed in the (i) date of first oral feeds (POD 7.7 ± 3.2 vs 16.1 ± 7.7: p ConclusionInitial feeding schedules with oral or incremental gavage-fed rates of at least 2.5 ml/kg in stepwise increments and multi-steps per day is wholly feasible in the postoperative feeding regimens of neonates with congenital duodenal disorders. Significant health benefits are thus achievable in these infants allowing an earlier time to acquiring full enteral feeding and their hospital discharge

Current treatment and outcomes of pediatric gastrointestinal stromal tumors (GIST): a systematic review of published studies

Gastrointestinal stromal tumor (GIST) is a rare cancer of mesenchymal origin mostly seen in adult and elderly populations. Therefore, the prognostic and therapeutic features of pediatric GIST are not clearly defined. Clinical knowledge has been largely extrapolated from case series and adult studies. In this systematic review, we aimed to analyze the health outcome metrics of pediatric GIST. Medline and Embase databases were searched using relevant key terms. The original search retrieved 1,892 titles; 27 studies with 184 patients (68% female) were included for final review. The primary tumors were located in the stomach (165/184, 90%), small bowel (12/184, 7%), and elsewhere (7/184, 4%). Individual patient data were available in 125 cases with a median follow-up of 6.7 years. All patients underwent surgical resection, which varied from wide local excision to total gastrectomy. There were 12 deaths (10%), 65 (52%) patients were alive with no evidence of disease, and 31 cases (25%) were alive with disease. Tumor size > 5 cm, high mitotic index, and spindle morphology were predictive of mortality. Pediatric GIST has a more favorable prognosis and different characteristics versus adult tumors. There is a crucial need for international consensus and specific pediatric guidelines for the treatment of this rare tumor

Stage 4S Neuroblastoma: What Are the Outcomes? A Systematic Review of Published Studies

Introduction The prognosis of stage 4S/MS neuroblastoma has traditionally been reported as excellent, yet conflicting treatment protocols exist for this enigmatic disease. To critically address this question, we have undertaken a systematic review of published studies to accurately determine outcomes for infants with stage 4S/MS neuroblastoma.Materials and Methods Studies were identified using MEDLINE, Embase, and Cochrane databases using the relevant search terms. Literature reviews, case reports, and adult studies were excluded. Data were extracted independently following article selection by three authors and reviewed by the senior author.Results The original search retrieved 2,325 articles. Following application of exclusion criteria and removing duplicate data, 37 studies (1,105 patients) were included for final review. Overall patient survival was 84%. Twelve studies (544 patients) recorded MYCN status. Mortality in MYCN amplified tumors was 56%. Chromosome 1p/11q status was reported in four studies and 1p/11q deletion carried a 40% fatality rate. Management included observation only (201 patients, 8.5% mortality), surgical resection of primary tumor only (153 patients, 6.5% mortality), chemotherapy only (186 patients, 21% mortality), radiotherapy (5 deaths, 33% mortality), chemotherapy with surgery (160 patients, 10% mortality), surgery with radiotherapy (21 patients, 19% mortality), radiotherapy with chemotherapy (42 patients, 29% mortality), and surgery with chemotherapy and radiotherapy (27 patients, 33% mortality).Conclusion There is a significant mortality observed in stage 4S/MS neuroblastoma infants with a dismal outcome observed in those patients with MYCN amplification and 1p/11q deletion. Those patients suitably amenable for conservative management or surgery to excise the primary tumor carry the best prognosis.</p

Neuroblastoma: the association of anatomical tumour site, molecular biology and patient outcomes

Background Numerous factors have been identified as carrying prognostic value in neuroblastoma (NB) and therefore incorporated in risk stratification of disease. Here, we investigate the association of anatomical site of NB with molecular biology and clinical outcomes.Methods A total of 117 patients with NB were studied over a 30-year period. Tumour location was confirmed with computed tomography/magnetic resonance imaging. Data on molecular biology were obtained as testing became available. Chi-squared, Fisher's exact test and Kaplan-Meier log-rank tests were used for statistical analysis.Results Tumour originated in the thoracic region (thoracic NB, TNB) in 15 patients (13%), adrenal gland (adrenal NB, ANB) in 88 patients (75%) and abdominal/paravertebral chain (paravertebral NB, PVNB) in 14 patients (12%). Overall survival (OS) for ANB was significantly lower (38%; P = 0.015). ANB cases were more frequently diagnosed at stage IV (69%; P = 0.001). MYCN amplification was noted in 33% of ANB cases and associated with lower OS (17% versus 62% MYCN non-amplified ANB; P = 0.01). The vast majority of TNB and PVNB were non-MYCN amplified (100% and 86%, respectively) and carried better prognosis (OS 86% and 83%, respectively). Forty-two percent of ANB cases were diploid and had lower OS (20% versus 71% hyperdiploid ANB; P = 0.079). TNB and PVNB were found to be mostly hyperdiploid (86% and 100%, respectively) with better OS (83% and 33%, respectively). Segmental chromosomal alterations had prognostic significance in those with PVNB (P = 0.03).Conclusion TNB tumours have better outcomes than adrenal tumours. This may be due to varied factors reported here including non-metastatic disease at presentation, non-amplification of the MYCN oncogene and overall favourable molecular biology characteristics