Extended induction chemotherapy does not improve the outcome for high-risk neuroblastoma patients: results of the randomized open-label GPOH trial NB2004-HR.

BACKGROUND: Long-term survival of high-risk neuroblastoma patients is still below 50% despite intensive multimodal treatment. This trial aimed to address whether the addition of two topotecan-containing chemotherapy courses compared to standard induction therapy improves event-free survival (EFS) of these patients. PATIENTS AND METHODS: An open-label, multicenter, prospective randomized controlled trial was carried out at 58 hospitals in Germany and Switzerland. Patients aged 1-21 years with stage 4 neuroblastoma and patients aged 6 months to 21 years with MYCN-amplified tumors were eligible. The primary endpoint was EFS. Patients were randomly assigned to standard induction therapy with six chemotherapy courses or to experimental induction chemotherapy starting with two additional courses of topotecan, cyclophosphamide, and etoposide followed by standard induction chemotherapy (eight courses in total). After induction chemotherapy, all patients received high-dose chemotherapy with autologous hematopoietic stem cell rescue and isotretinoin for consolidation. Radiotherapy was applied to patients with active tumors at the end of induction chemotherapy. RESULTS: Of 536 patients enrolled in the trial, 422 were randomly assigned to the control arm (n = 211) and the experimental arm (n = 211); the median follow-up time was 3.32 years (interquartile range 1.65-5.92). At data lock, the 3-year EFS of experimental and control patients was 34% and 32% [95% confidence Interval (CI) 28% to 40% and 26% to 38%; P = 0.258], respectively. Similarly, the 3-year overall survival of the patients did not differ [54% and 48% (95% CI 46% to 62% and 40% to 56%), respectively; P = 0.558]. The response to induction chemotherapy was not different between the arms. The median number of non-fatal toxicities per patient was higher in the experimental group while the median number of toxicities per chemotherapy course was not different. CONCLUSION: While the burden for the patients was increased by prolonging the induction chemotherapy and the toxicity, the addition of two topotecan-containing chemotherapy courses did not improve the EFS of high-risk neuroblastoma patients and thus cannot be recommended. CLINICAL TRIALS. GOV NUMBER: NCT number 03042429.

Comprehensive characterization of RB1 mutant and MYCN amplified retinoblastoma cell lines.

In retinoblastoma research tumor-derived cell lines remain an important model to investigate tumorigenesis and new therapy options, due to limited tumor material and lack of adequate animal models. A panel of 10 retinoblastoma cell lines was characterized with respect to mutation, methylation and expression of RB1 and MYCN. These established retinoblastoma cell lines represent the most frequent types of RB1 inactivation and together with the MYCN amplification status, three classes can be distinguished: RB1mut/MYCNnonA, RB1mut/MYCNA and RB1wt/MYCNA. MYCN amplification was identified in five cell lines, whereby two of them, RB522 and RB3823, harbor no aberration in RB1. Targeted sequencing of 160 genes often mutated in cancer identified only few variants in tumor-associated genes other than in RB1. None of these variants was recurrent. mRNA expression analyses of retinal markers, cell cycle regulators and members of the TP53 signaling pathway revealed a high variability between cell lines but no class-specific differences. The here presented thorough validation of retinoblastoma cell lines, including microsatellite analysis for cell line authentication, provides the basis for further in vitro studies on retinoblastoma.

Hox-C9 activates the intrinsic pathway of apoptosis and is associated with spontaneous regression in neuroblastoma.

Neuroblastoma is an embryonal malignancy of the sympathetic nervous system. Spontaneous regression and differentiation of neuroblastoma is observed in a subset of patients, and has been suggested to represent delayed activation of physiologic molecular programs of fetal neuroblasts. Homeobox genes constitute an important family of transcription factors, which play a fundamental role in morphogenesis and cell differentiation during embryogenesis. In this study, we demonstrate that expression of the majority of the human HOX class I homeobox genes is significantly associated with clinical covariates in neuroblastoma using microarray expression data of 649 primary tumors. Moreover, a HOX gene expression-based classifier predicted neuroblastoma patient outcome independently of age, stage and MYCN amplification status. Among all HOX genes, HOXC9 expression was most prominently associated with favorable prognostic markers. Most notably, elevated HOXC9 expression was significantly associated with spontaneous regression in infant neuroblastoma. Re-expression of HOXC9 in three neuroblastoma cell lines led to a significant reduction in cell viability, and abrogated tumor growth almost completely in neuroblastoma xenografts. Neuroblastoma growth arrest was related to the induction of programmed cell death, as indicated by an increase in the sub-G1 fraction and translocation of phosphatidylserine to the outer membrane. Programmed cell death was associated with the release of cytochrome c from the mitochondria into the cytosol and activation of the intrinsic cascade of caspases, indicating that HOXC9 re-expression triggers the intrinsic apoptotic pathway. Collectively, our results show a strong prognostic impact of HOX gene expression in neuroblastoma, and may point towards a role of Hox-C9 in neuroblastoma spontaneous regression.

MicroRNA miR-885-5p targets CDK2 and MCM5, activates p53 and inhibits proliferation and survival.

Several microRNA (miRNA) loci are found within genomic regions frequently deleted in primary neuroblastoma, including miR-885-5p at 3p25.3. In this study, we demonstrate that miR-885-5p is downregulated on loss of 3p25.3 region in neuroblastoma. Experimentally enforced miR-885-5p expression in neuroblastoma cell lines inhibits proliferation triggering cell cycle arrest, senescence and/or apoptosis. miR-885-5p leads to the accumulation of p53 protein and activates the p53 pathway, resulting in upregulation of p53 targets. Enforced miR-885-5p expression consistently leads to downregulation of cyclin-dependent kinase (CDK2) and mini-chromosome maintenance protein (MCM5). Both genes are targeted by miR-885-5p via predicted binding sites within the 3'-untranslated regions (UTRs) of CDK2 and MCM5. Transcript profiling after miR-885-5p introduction in neuroblastoma cells reveals alterations in expression of multiple genes, including several p53 target genes and a number of factors involved in p53 pathway activity. Taken together, these data provide evidence that miR-885-5p has a tumor suppressive role in neuroblastoma interfering with cell cycle progression and cell survival.

Integrated genomic profiling identifies two distinct molecular subtypes with divergent outcome in neuroblastoma with loss of chromosome 11q.

Imbalances in chromosome 11q occur in approximately 30% of primary neuroblastoma and are associated with poor outcome. It has been suggested that 11q loss constitutes a distinct clinico-genetic neuroblastoma subgroup by affecting expression levels of corresponding genes. This study analysed the relationship of 11q loss, clinical phenotype and global transcriptomic profiles in four clinico-genetic subgroups (11q alteration/favourable outcome, n=7; 11q alteration/unfavourable outcome, n=14; no 11q alteration/favourable outcome, n=81; no 11q alteration/unfavourable outcome, n=8; tumours with MYCN amplification and/or 1p loss were excluded). Unsupervised and supervised comparisons of gene expression profiles consistently showed significantly different mRNA patterns between favourable and unfavourable neuroblastomas, both in the subgroups with and without 11q loss. In contrast, favourable tumours with and without 11q loss showed highly similar transcriptomic profiles. Disproportionate downregulation of 11q genes was observed only in unfavourable tumours with 11q loss. The diverging molecular profiles were neither caused by considerable differences in the size of the deleted regions nor by differential methylation patterns of 11q genes. Together, this study shows that neuroblastoma with 11q loss comprises two biological subgroups that differ both in their clinical phenotype and gene expression patterns, indicating that 11q loss is not a primary determinant of neuroblastoma tumour behaviour.

IgE-mediated hypersensitivity to latex in childhood.

A total of 267 children scheduled to receive anesthesia during a surgical, neurosurgical, or orthopedic intervention were investigated. IgE antibodies against latex were detected in serum samples of 6.4% (17/267 children) of the patients. The most important difference between sensitized and nonsensitized children was the number of surgical interventions in the past. The median of surgical interventions was 1.0 in the nonsensitized group of children and 3.0 in the sensitized group. Only 0.9% of the children with up to two surgical interventions and 34.1% with three or more procedures were sensitized to latex. Only one of the sensitized children developed intraoperative anaphylaxis during intervention after our investigation. We conclude that children with a history of three or more surgical interventions have a high risk of sensitization to latex proteins. Nevertheless, the predictive value of IgE antibodies against latex for development of anaphylaxis during anesthesia seems to be low.

The effect of general anaesthesia on breathing patterns in elderly patients during the early post-operative period.

This study was designed to investigate the incidence of critical events in breathing following light general anesthesia compared with normal sleep during the first 12 h after transfer from the recovery room. There were no significant differences in the incidence of apnoea or desaturation episodes between normal sleep and the post-operative recovery period. There was a close correlation between the pre-operative and post-operative incidence of apnoea (r = 0.93), pre-operative and post-operative desaturation periods (r = 0.81), pre-operative and post-operative mean SpO2 values (r = 0.54) and pre-operative and post-operative minimal SpO2 values (r = 0.90) in all the patients. In the early post-operative period, breathing patterns and oxygenation were similar to those observed during normal night-time sleep in elderly patients undergoing ophthalmological surgery.

Pulmonary blood flow regulation: influence of positive pressure ventilation.

Positive pressure ventilation with PEEP has been reported to reduce hypoxic pulmonary vasoconstriction (HPV) and thus increase venous admixture. This effect was investigated in six chronically instrumented unanesthetized healthy sheep. The change in left pulmonary arterial blood flow (Qlpa, ultrasonic transit time) in response to unilateral lung hypoxia (10 min of N2 alternately to the left (LLH) and right lung (RLH) was evaluated during mechanical ventilation with and without PEEP of 10 cm H2O in comparison to spontaneous breathing. In the spontaneously breathing animal and during mechanical ventilation without PEEP, Qlpa decreased during LLH from 30% to 16% of cardiac index, during RLH it increased to 51%. With 10 cm H2O of PEEP, Qlpa showed an identical reaction to hypoxia both in the left and right lung. It is concluded that mechanical ventilation and PEEP up to 10 cm H2O does not interfere with pulmonary blood flow regulation to hypoxia.

[Allergic and pseudo-allergic reactions in anesthesia. II: Symptoms, diagnosis, therapy, prevention]. / Allergische und pseudoallergische Reaktionen in der Anästhesie. Teil II: Symptomatik, Diagnose, Therapie, Prophylaxe.

In this article we present the symptomatic features and discuss relevant diagnostic and therapeutic aspects of anaphylactoid reactions. In addition we give practical advice as to how to avoid and manage allergic or pseudoallergic reactions during anaesthesia. Measurements of serum tryptase-levels and of methylhistamine in urine have been introduced in clinical diagnostic routine. Skin tests and determination of specific antibodies are essential to identify responsible substances. Preventive measures like careful premedication, calm atmosphere, slow injection of drugs, the use of diluted solutions, and the use of drugs with a low potential for anaphylactoid reactions are important. Substances like inhalation anaesthetics, propofol, etomidate, ketamine, midazolam, fentanyl, alfentanil and bupivacain without epinephrine should be used.

[Allergic and pseudo-allergic reactions in anesthesia. I: Pathogenesis, risk factors, substances]. / Allergische und pseudoallergische Reaktionen in der Anästhesie. Teil I: Pathogenese, Risikofaktoren, Substanzen.

In this article we present a survey on the pathogenesis of allergic or pseudoallergic reactions in anaesthesia, the risk factors and the responsible substances. The incidence of anaphylactoid reactions is between 1:3500 and 1:20,000 anaesthetic cases. The estimated mortality rate amounts to 3-6%. Neuromuscular blocking drugs account for most of the cases of significant anaphylactoid reactions (59-70%). However, the incidence of latex-related reactions in increasing. Risk factors for anaphylaxis are a history of IgE-mediated drug allergy, repeated anaesthesias, atopy, hyperventilation tetany, and the use of neuromuscular blocking drugs in females. Risk factors for pseudoallergic reactions are emotional stress, atopic predisposition, increased sensitivity for histamin, hyperventilation tetany and female gender.

[Hemo- and cardiodynamic effect of nifedipine in halothane or isoflurane anesthesia. An animal experiment study]. / Hämo- und kardiodynamische Wirkung von Nifedipin unter Halothan- oder Isoflurananästhesie. Eine tierexperimentelle Studie.

OBJECTIVE: The present experimental study on 16 acutely instrumented dogs was designed to determine the haemo- and cardiodynamic changes after an intravenous infusion of nifedipine during halothane or isoflurane anaesthesia. METHODS: General anaesthesia was induced with ketamine (10 mg/kg) and fentanyl (0.02 mg/kg) and maintained with fentanyl (0.3 micrograms/kg/min), 3:1 N2O/O2 inhalation mixture, and pancuronium (300 micrograms/kg/h). A left thoracotomy was performed and a needle force probe was placed in the left ventricular wall to measure myocardial force of contraction. A Widney gauge was placed around the left ventricle to measure left ventricular circumference changes. The animals were also monitored with left ventricular tip manometers, pulmonary arterial thermodilution catheters, and femoral arterial and venous catheters. Three hours after instrumentation baseline haemodynamic measurements were performed and repeated 30 min after either halothane 0.8 vol.% (n = 8) or isoflurane 1.5 vol.% (n = 8). Then nifedipine (10 micrograms/kg i.v.) was administered and haemodynamic measurements were repeated. RESULTS: Both volatile anaesthetic agents caused a decrease in MAP, CO, LVP, LVFS, and dP/dtmax. Heart rate, CVP, PAOP, and the diastolic diameter of the heart did not change with halothane and isoflurane. Isoflurane led to a decrease of SVR that was not seen with the administration of halothane. Nifedipine during halothane anaesthesia caused a further decrease in MAP, SVR, LVP, dP/dtmax, and LVFS compared to the already reduced values with halothane alone. However, SV did not decrease any further. If nifedipine was added to isoflurane a further decrease in CO and SV was observed despite a constant SVR. CONCLUSION: Halothane, isoflurane and nifedipine are cardiac depressant drugs. Isoflurane induces vasodilation and appears to be less cardiodepressant than halothane in the clinical situation. However, if nifedipine is added, the vasodilation caused by nifedipine offsets its own negative inotropic effect and in parts the cardiac depression of halothane. Combined with isoflurane the vasodilatory effect of nifedipine is insignificant and the negative inotropic effects of both drugs are additive resulting in a profound decrease in SV and CO.

[Eicosanoids as mediators in ARDS]. / Die Eicosanoide als Mediatoren beim ARDS.

Eicosanoids partly develop from the metabolism of arachidonic acid through the cyclooxygenase or the lipoxygenase pathway. Lipoxygenase products are the leukotrienes (LTA4, LTB4, LTC4, LTD4, LTE4) and the 5-hydroxyeicosatetraenoic acid (5-HETE). Cyclooxygenase products are the prostanoids (prostaglandins [PG] D2, E2, F2, I2 and thromboxane A2). The other part of the eicosanoids develops from the metabolism of two other fatty acids over the same pathways; 8,11,14-eicosatrienoic acid leads to the prostaglandins D1, E1, F1, I1 and the leukotrienes A3, B3, C3, D3, E3. From 5,8,11,14,17-eicosapentaenoic acid result the prostaglandins D3, E3, F3, I3 and the leukotrienes A5, B5, C5, D5, E5. The pathophysiological changes in ARDS are mainly due to an imbalance of opposing effects of mediators. In this regard eicosanoids play an important role which has not yet been clearly determined. Bronchoconstriction and pulmonary hypertension are increased by thromboxane A2 and leukotrienes, whereas they are reduced by PGI2. Pulmonary edema is enlarged by leukotriene, especially, LTB4, whereas PGI2 has a protective effect. The aggregation of platelets is mediated through thromboxane A2, PGF2 and LTB4; PGE1 and PGI2 counteract these reactions. LTB4, in addition to 5-HETE, leads to the activation of inflammatory cells. Drug induced eicosanoid imbalances can be used for therapeutic interventions.

[An anaphylactic reaction under anesthesia due to a combined latex and ethylene oxide allergy]. / Anaphylaktische Reaktion unter Narkose aufgrund einer kombinierten Latex- und Athylenoxidallergie.

There is an increasing number of reports on anaphylactic reactions due to latex. We report on a 9-year-old boy who developed anaphylaxis shortly after induction of anaesthesia due to a combined latex and ethylene oxide allergy. Patients with multiple medical treatment and those with atopy seem to be at risk.

Ventilation with positive end-expiratory airway pressure causes leukocyte retention in human lung.

We examined the effect of assisted ventilation with different positive end-expiratory airway pressures (PEEP) on pulmonary leukocyte retention in humans after cardiopulmonary bypass. Eight patients who underwent heart surgery were ventilated in the postoperative phase briefly with 10 (39.6 +/- 0.9 s) and 20 cmH2O (40.3 +/- 1.0 s) PEEP. Before, during, and after this ventilatory maneuver, blood was withdrawn simultaneously from catheters placed in the pulmonary and radial arteries for blood cell differentials. At the same time points, pulmonary and systemic hemodynamics were recorded. During PEEP ventilation, there was a four- (10 cmH2O PEEP) and an eightfold (20 cmH2O PEEP) increase in mixed venous-arterial leukocyte cell difference compared with baseline. This phenomenon was based mainly on a transpulmonary cell difference of polymorphonuclear cells. Likewise, the lymphocytes were entrapped in the pulmonary vasculature during PEEP ventilation. During the ventilatory maneuver, the pulmonary blood flow was significantly reduced; it was indexed by a declined cardiac output. We conclude that PEEP ventilation in the post-operative phase after cardiopulmonary bypass causes pulmonary polymorphonuclear cell entrapment. The most likely mechanism for this phenomenon is the compression of alveolar capillaries and reduced pulmonary blood flow in response to the raised alveolar pressure.

The influence of different procedures of general anaesthesia on platelet function, coagulation and the fibrinolytic system.

We investigated whether different procedures during general anaesthesia alter platelet activation in vivo and/or activate coagulation and fibrinolysis. Forty-one healthy adult patients, scheduled for elective ophthalmic surgery under general anaesthesia, were studied with regard to changes of plasma beta-thromboglobulin (beta TG, index of platelet activation), thrombin-antithrombin III-complex (TAT, index of activation of coagulation) and d-dimer (index of fibrinolysis) during anaesthesia. The patients underwent either inhalation anaesthesia with enflurane and nitrous oxide or balanced anaesthesia with enflurane (0.5% end-tidal concentration) and alfentanil. Ten minutes after intubation the beta TG level was significantly reduced compared to the preoperative value in both general anaesthesia groups. Balanced anaesthesia caused a moderate but significant increase of TAT values at 10 min after extubation. No significant change in d-dimer levels was seen. Presuming a minimal effect of the surgical procedure on the determined variables, we conclude that none of the anaesthetic procedures induces platelet activation and fibrinolysis. The clinical relevance of the moderate coagulation activation during balanced anaesthesia remains to be investigated.

Carbon monoxide and pulmonary circulation in an ovine model.

The direct pulmonary vasoconstrictive effects of inhaled carbon monoxide were evaluated in chronically instrumented and anesthetized sheep (1.7% halothane in air) (n = 8). The response to carbon monoxide (2%), which was applied for 8 minutes through a double-lumen tube alternately to the left or right lung of each animal, was compared with baseline values. The induced carboxyhemoglobin level (65%) led to increases in cardiac output, pulmonary arterial pressure, stroke volume index, and heart rate. Systemic vascular resistance decreased, and pulmonary vascular resistance and mean arterial pressure were unchanged. The changes in pressure and flow were equivalent no matter which lung was exposed to carbon monoxide. No diversion of blood from one lung to the other was observed during the test period. We conclude that carbon monoxide does not have a direct pulmonary vasoconstrictive effect. The increase in pulmonary arterial pressure is a result of the decrease in mixed venous oxygen content (stimulus for hypoxic pulmonary vasoconstriction) and the increase in cardiac output.