Clinical Phenotype and Management of Severe Neurotoxicity Observed in Patients with Neuroblastoma Treated with Dinutuximab Beta in Clinical Trials.

Neurotoxicity is an off-tumour, on-target side effect of GD2-directed immunotherapy with monoclonal antibodies. Here, we report the frequency, management and outcome of patients enrolled in two prospective clinical trials who experienced severe neurotoxicity during immunotherapy with the anti-GD2 antibody dinutuximab beta (DB) administered as short-term infusion (HR-NBL1/SIOPEN study, randomisation R2, EudraCT 2006-001489-17) or as long-term infusion (HR-NBL1/SIOPEN study, randomisation R4, EudraCT 2006-001489-17 and LTI/SIOPEN study, EudraCT 2009-018077-31), either alone or with subcutaneous interleukin-2 (scIL-2). The total number of patients included in this analysis was 1102. Overall, 44/1102 patients (4.0%) experienced Grade 3/4 neurotoxicities (HR-NBL1 R2, 21/406; HR-NBL1 R4, 8/408; LTI study, 15/288), including 27 patients with severe neurotoxicities (2.5%). Events occurred predominantly in patients receiving combined treatment with DB and scIL-2. Neurotoxicity was treated using dexamethasone, prednisolone, intravenous immunoglobulins and, in two patients, plasmapheresis, which was highly effective. While neurological recovery was observed in 16 of 21 patients with severe neurotoxicities, 5/1102 (0.45%) patients experienced persistent and severe neurological deficits. In conclusion, severe neurotoxicity is most commonly observed in patients receiving DB with scIL-2. Considering the lack of clinical benefit for IL-2 in clinical trials so far, the administration of IL-2 alongside DB is not recommended.

Age Dependency of the Prognostic Impact of Tumor Genomics in Localized Resectable MYCN-Nonamplified Neuroblastomas. Report From the SIOPEN Biology Group on the LNESG Trials and a COG Validation Group.

PURPOSE: For localized, resectable neuroblastoma without MYCN amplification, surgery only is recommended even if incomplete. However, it is not known whether the genomic background of these tumors may influence outcome. PATIENTS AND METHODS: Diagnostic samples were obtained from 317 tumors, International Neuroblastoma Staging System stages 1/2A/2B, from 3 cohorts: Localized Neuroblastoma European Study Group I/II and Children's Oncology Group. Genomic data were analyzed using multi- and pangenomic techniques and fluorescence in-situ hybridization in 2 age groups (cutoff age, 18 months) and were quality controlled by the International Society of Pediatric Oncology European Neuroblastoma (SIOPEN) Biology Group. RESULTS: Patients with stage 1 tumors had an excellent outcome (5-year event-free survival [EFS] ± standard deviation [SD], 95% ± 2%; 5-year overall survival [OS], 99% ± 1%). In contrast, patients with stage 2 tumors had a reduced EFS in both age groups (5-year EFS ± SD, 84% ± 3% in patients < 18 months of age and 75% ± 7% in patients ≥ 18 months of age). However, OS was significantly decreased only in the latter group (5-year OS ± SD in < 18months and ≥ 18months, 96% ± 2% and 81% ± 7%, respectively; P = .001). In < 18months, relapses occurred independent of segmental chromosome aberrations (SCAs); only 1p loss decreased EFS (5-year EFS ± SD in patients 1p loss and no 1p loss, 62% ± 13% and 87% ± 3%, respectively; P = .019) but not OS (5-year OS ± SD, 92% ± 8% and 97% ± 2%, respectively). In patients ≥ 18 months, only SCAs led to relapse and death, with 11q loss as the strongest marker (11q loss and no 11q loss: 5-year EFS ± SD, 48% ± 16% and 85% ± 7%, P = .033; 5-year OS ± SD, 46% ± 22% and 92% ± 6%, P = .038). CONCLUSION: Genomic aberrations of resectable non-MYCN-amplified stage 2 neuroblastomas have a distinct age-dependent prognostic impact. Chromosome 1p loss is a risk factor for relapse but not for diminished OS in patients < 18 months, SCAs (especially 11q loss) are risk factors for reduced EFS and OS in those > 18months. In older patients with SCA, a randomized trial of postoperative chemotherapy compared with observation alone may be indicated.

Ch14.18 antibody produced in CHO cells in relapsed or refractory Stage 4 neuroblastoma patients: a SIOPEN Phase 1 study.

PURPOSE: This study aimed to assess the safety, pharmacokinetic and activity profiles of the human-mouse chimeric monoclonal anti-disialoganglioside GD2 antibody ch14.18 produced in Chinese hamster ovary (CHO) cells (ch14.18/CHO). METHODS: Sixteen children with recurrent/refractory neuroblastoma (median age 7.6 y) were enrolled in this Phase 1 dose-finding study. Patients received ch14.18/CHO courses of 10, 20 or 30 mg/m (2)/day as an eight-hour infusion over five consecutive days. Three courses at the same dose level were allowed unless disease progressed. Clearance and biodistribution of radiolabelled ch14.18/CHO in Balb/c and A/J mice were analyzed. RESULTS: A total of 41 ch14.18/CHO courses were given (10 × 3 courses, 5 × 2 courses, 1 × 1 course). Side effects were similar in expectedness, frequency and magnitude to those reported for ch14.18/SP2/0. The dose level of 20 mg/m(2)/day was confirmed. Toxicity was reversible and no treatment-related deaths occurred. In children, the peak plasma concentration was 16.51 µg/ml ± 5.9 µg/ml and the half-life was 76.91 h ± 52.5 h. A partial response following ch14.18/CHO was observed in 2/7 patients with residual disease. In mice, the half-lives were 22.7 h ± 1.9h for ch14.18/CHO and 25.0 h ± 1.9 h for ch14.18/SP2/0. The biodistribution of (125)I-ch14.18/CHO in mice with neuroblastoma was identical to (125)I-ch14.18/SP2/0, indicating GD 2 targeting activity in vivo. Ch14.18 produced in CHO cells showed an unchanged toxicity profile and pharmacokinetics in neuroblastoma patients compared with ch14.18 produced in SP2/0 cells, and evidence of clinical activity was observed. In mice, analysis of pharmacokinetics and biodistribution showed comparable results between ch14.18/CHO and ch14.18/SP2/0. Based on these results, ch14.18/CHO was accepted for prospective clinical evaluation.

CD1 gene polymorphisms and phenotypic variability in X-linked adrenoleukodystrophy.

X-linked adrenoleukodystrophy (X-ALD) is characterized by marked phenotypic variation ranging from adrenomyeloneuropathy (AMN) to childhood cerebral ALD (CCALD). X-ALD is caused by mutations in the ABCD1 gene, but no genotype-phenotype correlation has been established so far and modifier gene variants are suspected to modulate phenotypes. Specific classes of lipids, enriched in very long-chain fatty acids that accumulate in plasma and tissues from X-ALD patients are suspected to be involved in the neuroinflammatory process of CCALD. CD1 proteins are lipid- antigen presenting molecules encoded by five CD1 genes in human (CD1A-E). Association studies with 23 tag SNPs covering the CD1 locus was performed in 52 patients with AMN and 87 patients with CCALD. The minor allele of rs973742 located 4-kb downstream from CD1D was significantly more frequent in AMN patients (χ²â€Š= 7.6; P = 0.006). However, this association was no longer significant after Bonferroni correction for multiple testing. The other polymorphisms of the CD1 locus did not reveal significant association. Further analysis of other CD1D polymorphisms did not detect stronger association with X-ALD phenotypes. Although the association with rs973742 warrants further investigations, these results indicate that the genetic variants of CD1 genes do not contribute markedly to the phenotypic variance of X-ALD.

Acyl-CoA synthetase activity links wild-type but not mutant alpha-synuclein to brain arachidonate metabolism.

Because alpha-synuclein (Snca) has a role in brain lipid metabolism, we determined the impact that the loss of alpha-synuclein had on brain arachidonic acid (20:4n-6) metabolism in vivo using Snca-/- mice. We measured [1-(14)C]20:4n-6 incorporation and turnover kinetics in brain phospholipids using an established steady-state kinetic model. Liver was used as a negative control, and no changes were observed between groups. In Snca-/- brains, there was a marked reduction in 20:4n-6-CoA mass and in microsomal acyl-CoA synthetase (Acsl) activity toward 20:4n-6. Microsomal Acsl activity was completely restored after the addition of exogenous wild-type mouse or human alpha-synuclein, but not by A30P, E46K, and A53T forms of alpha-synuclein. Acsl and acyl-CoA hydrolase expression was not different between groups. The incorporation and turnover of 20:4n-6 into brain phospholipid pools were markedly reduced. The dilution coefficient lambda, which indicates 20:4n-6 recycling between the acyl-CoA pool and brain phospholipids, was increased 3.3-fold, indicating more 20:4n-6 was entering the 20:4n-6-CoA pool from the plasma relative to that being recycled from the phospholipids. This is consistent with the reduction in Acsl activity observed in the Snca-/- mice. Using titration microcalorimetry, we determined that alpha-synuclein bound free 20:4n-6 (Kd = 3.7 microM) but did not bind 20:4n-6-CoA. These data suggest alpha-synuclein is involved in substrate presentation to Acsl rather than product removal. In summary, our data demonstrate that alpha-synuclein has a major role in brain 20:4n-6 metabolism through its modulation of endoplasmic reticulum-localized acyl-CoA synthetase activity, although mutant forms of alpha-synuclein fail to restore this activity.

Contribution of epigenetic silencing of tumor necrosis factor-related apoptosis inducing ligand receptor 1 (DR4) to TRAIL resistance and ovarian cancer.

Dysregulation of apoptosis may support tumorigenesis by allowing cells to live beyond their normally intended life span. The various receptors for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) are located on chromosome 8p21.2, a region frequently deleted in ovarian cancer. Lack of expression of TRAIL receptor 1 (death receptor 4, DR4) correlates with resistance to TRAIL-induced apoptosis in ovarian cancer cells. Reconstitution of DR4 in the TRAIL-resistant A2780 ovarian cancer cell line was investigated with the demethylating agent 5-aza-2'-deoxycytidine and transient gene transfer. Regulation of other genes in the TRAIL pathway by 5-aza-2'-deoxycytidine was assessed in DNA GeneChip experiments. Primary ovarian cancers were analyzed by methylation-specific PCR and immunohistochemical analysis of a tissue microarray. Regulation of DR4 expression by demethylation or transient transfection is of functional relevance for TRAIL resistance in an ovarian cancer cell line. Hypermethylation of the DR4 promoter could be found in 10 of 36 (27.7%) DNAs isolated from ovarian cancer tissue. In an independent set of 68 ovarian cancer cases, a complete loss or down-regulation of DR4 protein expression was observed 10.3% and 8.8% patients, respectively. A significant (P = 0.019) majority of these patients was below 50 years of age. Our findings show a functional relevance of the level of DR4 expression in ovarian cancer and suggest a substantial contribution of DR4 hypermethylation and consequent loss of DR4 expression to ovarian cancer pathogenesis, particularly in premenopausal patients.

Resistance to TRAIL-induced apoptosis in ovarian cancer cell lines is overcome by co-treatment with cytotoxic drugs.

BACKGROUND: TRAIL, tumor necrosis factor-related apoptosis-inducing ligand, is a recently identified cytokine that preferentially kills transformed cells while sparing most normal cells. METHODS: We investigated the ability of TRAIL alone and TRAIL in combination with cytotoxic drugs to induce apoptosis in six ovarian cancer cell lines. To get some insight into the resistance to TRAIL, the expression of TRAIL receptors and selected downstream signaling elements was determined. RESULTS: TRAIL induced significant apoptosis (up to 80%) in three out of six ovarian cancer cell lines (MZ-26, CaOV-3, ES-2). In A2780 and A2780ADR cells, resistance to TRAIL-induced apoptosis correlated with their lack of DR4-expression. MZ-15 cells, which expressed the processed form of FLIP(L), p43 (FADD-like IL-1beta-converting enzyme (FLICE)-like inhibitory protein (FLIP)), and FLIP(S), were resistant to TRAIL in spite of the presence of DR4. When TRAIL-resistant cell lines were co-incubated with routinely used cytotoxic agents, TRAIL exerted a synergistic effect leading to apoptosis rates unachievable by incubation with cytotoxic agents alone. CONCLUSION: The ability of TRAIL to induce apoptosis in ovarian cancer cells as well as to potentiate the activity of chemotherapeutic agents even in cell lines that are resistant to TRAIL-induced cytotoxicity is a powerful promise in the fight against this deadly disease.