Wendy B. London

Anti-GD2 Antibody with GM-CSF, Interleukin-2, and Isotretinoin for Neuroblastoma

BACKGROUND Preclinical and preliminary clinical data indicate that ch14.18, a monoclonal antibody against the tumor-associated disialoganglioside GD2, has activity against neuroblastoma and that such activity is enhanced when ch14.18 is combined with granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-2. We conducted a study to determine whether adding ch14.18, GM-CSF, and interleukin-2 to standard isotretinoin therapy after intensive multimodal therapy would improve outcomes in high-risk neuroblastoma. METHODS Patients with high-risk neuroblastoma who had a response to induction therapy and stem-cell transplantation were randomly assigned, in a 1:1 ratio, to receive standard therapy (six cycles of isotretinoin) or immunotherapy (six cycles of isotretinoin and five concomitant cycles of ch14.18 in combination with alternating GM-CSF and interleukin-2). Event-free survival and overall survival were compared between the immunotherapy group and the standard-therapy group, on an intention-to-treat basis. RESULTS A total of 226 eligible patients were randomly assigned to a treatment group. In the immunotherapy group, a total of 52% of patients had pain of grade 3, 4, or 5, and 23% and 25% of patients had capillary leak syndrome and hypersensitivity reactions, respectively. With 61% of the number of expected events observed, the study met the criteria for early stopping owing to efficacy. The median duration of follow-up was 2.1 years. Immunotherapy was superior to standard therapy with regard to rates of event-free survival (66±5% vs. 46±5% at 2 years, P=0.01) and overall survival (86±4% vs. 75±5% at 2 years, P=0.02 without adjustment for interim analyses). CONCLUSIONS Immunotherapy with ch14.18, GM-CSF, and interleukin-2 was associated with a significantly improved outcome as compared with standard therapy in patients with high-risk neuroblastoma. (Funded by the National Institutes of Health and the Food and Drug Administration; ClinicalTrials.gov number, NCT00026312.)

The International Neuroblastoma Risk Group (INRG) Classification System: An INRG Task Force Report

PURPOSE Because current approaches to risk classification and treatment stratification for children with neuroblastoma (NB) vary greatly throughout the world, it is difficult to directly compare risk-based clinical trials. The International Neuroblastoma Risk Group (INRG) classification system was developed to establish a consensus approach for pretreatment risk stratification. PATIENTS AND METHODS The statistical and clinical significance of 13 potential prognostic factors were analyzed in a cohort of 8,800 children diagnosed with NB between 1990 and 2002 from North America and Australia (Childrens Oncology Group), Europe (International Society of Pediatric Oncology Europe Neuroblastoma Group and German Pediatric Oncology and Hematology Group), and Japan. Survival tree regression analyses using event-free survival (EFS) as the primary end point were performed to test the prognostic significance of the 13 factors. RESULTS Stage, age, histologic category, grade of tumor differentiation, the status of the MYCN oncogene, chromosome 11q status, and DNA ploidy were the most highly statistically significant and clinically relevant factors. A new staging system (INRG Staging System) based on clinical criteria and tumor imaging was developed for the INRG Classification System. The optimal age cutoff was determined to be between 15 and 19 months, and 18 months was selected for the classification system. Sixteen pretreatment groups were defined on the basis of clinical criteria and statistically significantly different EFS of the cohort stratified by the INRG criteria. Patients with 5-year EFS more than 85%, more than 75% to < or = 85%, > or = 50% to < or = 75%, or less than 50% were classified as very low risk, low risk, intermediate risk, or high risk, respectively. CONCLUSION By defining homogenous pretreatment patient cohorts, the INRG classification system will greatly facilitate the comparison of risk-based clinical trials conducted in different regions of the world and the development of international collaborative studies.

Activating mutations in ALK provide a therapeutic target in neuroblastoma.

Neuroblastoma, an embryonal tumour of the peripheral sympathetic nervous system, accounts for approximately 15% of all deaths due to childhood cancer. High-risk neuroblastomas are rapidly progressive; even with intensive myeloablative chemotherapy, relapse is common and almost uniformly fatal. Here we report the detection of previously unknown mutations in the ALK gene, which encodes a receptor tyrosine kinase, in 8% of primary neuroblastomas. Five non-synonymous sequence variations were identified in the kinase domain of ALK, of which three were somatic and two were germ line. The most frequent mutation, F1174L, was also identified in three different neuroblastoma cell lines. ALK complementary DNAs encoding the F1174L and R1275Q variants, but not the wild-type ALK cDNA, transformed interleukin-3-dependent murine haematopoietic Ba/F3 cells to cytokine-independent growth. Ba/F3 cells expressing these mutations were sensitive to the small-molecule inhibitor of ALK, TAE684 (ref. 4). Furthermore, two human neuroblastoma cell lines harbouring the F1174L mutation were also sensitive to the inhibitor. Cytotoxicity was associated with increased amounts of apoptosis as measured by TdT-mediated dUTP nick end labelling (TUNEL). Short hairpin RNA (shRNA)-mediated knockdown of ALK expression in neuroblastoma cell lines with the F1174L mutation also resulted in apoptosis and impaired cell proliferation. Thus, activating alleles of the ALK receptor tyrosine kinase are present in primary neuroblastoma tumours and in established neuroblastoma cell lines, and confer sensitivity to ALK inhibition with small molecules, providing a molecular rationale for targeted therapy of this disease.

Long-Term Results for Children With High-Risk Neuroblastoma Treated on a Randomized Trial of Myeloablative Therapy Followed by 13-cis-Retinoic Acid: A Children's Oncology Group Study

UNLABELLED PURPOSE We assessed the long-term outcome of patients enrolled on CCG-3891, a high-risk neuroblastoma study in which patients were randomly assigned to undergo autologous purged bone marrow transplantation (ABMT) or to receive chemotherapy, and subsequent treatment with 13-cis-retinoic acid (cis-RA). PATIENTS AND METHODS Patients received the same induction chemotherapy, with random assignment (N = 379) to consolidation with myeloablative chemotherapy, total-body irradiation, and ABMT versus three cycles of intensive chemotherapy. Patients who completed consolidation without disease progression were randomly assigned to receive no further therapy or cis-RA for 6 months. Results The event-free survival (EFS) for patients randomly assigned to ABMT was significantly higher than those randomly assigned to chemotherapy; the 5-year EFS (mean +/- SE) was 30% +/- 4% versus 19% +/- 3%, respectively (P = .04). The 5-year EFS (42% +/- 5% v 31% +/- 5%) from the time of second random assignment was higher for cis-RA than for no further therapy, though it was not significant. Overall survival (OS) was significantly higher for each random assignment by a test of the log(-log(.)) transformation of the survival estimates at 5 years (P < .01). The 5-year OS from the second random assignment of patients who underwent both random assignments and who were assigned to ABMT/cis-RA was 59% +/- 8%; for ABMT/no cis-RA, it was 41% +/- 8% [corrected]; for continuing chemotherapy/cis-RA, it was 38% +/- 7%; and for chemotherapy/no cis-RA, it was 36% +/- 7%. CONCLUSION Myeloablative therapy and autologous hematopoietic cell rescue result in significantly better 5-year EFS than nonmyeloablative chemo therapy; neither myeloablative therapy with [corrected] autologous hematopoietic cell rescue nor cis-RA given after consolidation therapy significantly improved OS.

The genetic landscape of high-risk neuroblastoma

Neuroblastoma is a malignancy of the developing sympathetic nervous system that often presents with widespread metastatic disease, resulting in survival rates of less than 50%. To determine the spectrum of somatic mutation in high-risk neuroblastoma, we studied 240 affected individuals (cases) using a combination of whole-exome, genome and transcriptome sequencing as part of the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) initiative. Here we report a low median exonic mutation frequency of 0.60 per Mb (0.48 nonsilent) and notably few recurrently mutated genes in these tumors. Genes with significant somatic mutation frequencies included ALK (9.2% of cases), PTPN11 (2.9%), ATRX (2.5%, and an additional 7.1% had focal deletions), MYCN (1.7%, causing a recurrent p.Pro44Leu alteration) and NRAS (0.83%). Rare, potentially pathogenic germline variants were significantly enriched in ALK, CHEK2, PINK1 and BARD1. The relative paucity of recurrent somatic mutations in neuroblastoma challenges current therapeutic strategies that rely on frequently altered oncogenic drivers.

The International Neuroblastoma Risk Group (INRG) Staging System: An INRG Task Force Report

PURPOSE The International Neuroblastoma Risk Group (INRG) classification system was developed to establish a consensus approach for pretreatment risk stratification. Because the International Neuroblastoma Staging System (INSS) is a postsurgical staging system, a new clinical staging system was required for the INRG pretreatment risk classification system. METHODS To stage patients before any treatment, the INRG Task Force, consisting of neuroblastoma experts from Australia/New Zealand, China, Europe, Japan, and North America, developed a new INRG staging system (INRGSS) based on clinical criteria and image-defined risk factors (IDRFs). To investigate the impact of IDRFs on outcome, survival analyses were performed on 661 European patients with INSS stages 1, 2, or 3 disease for whom IDRFs were known. RESULTS In the INGRSS, locoregional tumors are staged L1 or L2 based on the absence or presence of one or more of 20 IDRFs, respectively. Metastatic tumors are defined as stage M, except for stage MS, in which metastases are confined to the skin, liver, and/or bone marrow in children younger than 18 months of age. Within the 661-patient cohort, IDRFs were present (ie, stage L2) in 21% of patients with stage 1, 45% of patients with stage 2, and 94% of patients with stage 3 disease. Patients with INRGSS stage L2 disease had significantly lower 5-year event-free survival than those with INRGSS stage L1 disease (78% +/- 4% v 90% +/- 3%; P = .0010). CONCLUSION Use of the new staging (INRGSS) and risk classification (INRG) of neuroblastoma will greatly facilitate the comparison of risk-based clinical trials conducted in different regions of the world.

Copy number variation at 1q21.1 associated with neuroblastoma

Common copy number variations (CNVs) represent a significant source of genetic diversity, yet their influence on phenotypic variability, including disease susceptibility, remains poorly understood. To address this problem in human cancer, we performed a genome-wide association study of CNVs in the childhood cancer neuroblastoma, a disease in which single nucleotide polymorphism variations are known to influence susceptibility. We first genotyped 846 Caucasian neuroblastoma patients and 803 healthy Caucasian controls at ∼550,000 single nucleotide polymorphisms, and performed a CNV-based test for association. We then replicated significant observations in two independent sample sets comprised of a total of 595 cases and 3,357 controls. Here we describe the identification of a common CNV at chromosome 1q21.1 associated with neuroblastoma in the discovery set, which was confirmed in both replication sets. This CNV was validated by quantitative polymerase chain reaction, fluorescent in situ hybridization and analysis of matched tumour specimens, and was shown to be heritable in an independent set of 713 cancer-free parent–offspring trios. We identified a previously unknown transcript within the CNV that showed high sequence similarity to several neuroblastoma breakpoint family (NBPF) genes and represents a new member of this gene family (NBPF23). This transcript was preferentially expressed in fetal brain and fetal sympathetic nervous tissues, and the expression level was strictly correlated with CNV state in neuroblastoma cells. These data demonstrate that inherited copy number variation at 1q21.1 is associated with neuroblastoma and implicate a previously unknown neuroblastoma breakpoint family gene in early tumorigenesis of this childhood cancer.

Evidence for an Age Cutoff Greater Than 365 Days for Neuroblastoma Risk Group Stratification in the Children's Oncology Group

PURPOSE In the Childrens Oncology Group, risk group assignment for neuroblastoma is critical for therapeutic decisions, and patients are stratified by International Neuroblastoma Staging System stage, MYCN status, ploidy, Shimada histopathology, and diagnosis age. Age less than 365 days has been associated with favorable outcome, but recent studies suggest that older age cutoff may improve prognostic precision. METHODS To identify the optimal age cutoff, we retrospectively analyzed data from the Pediatric Oncology Group biology study 9047 and Childrens Cancer Group studies 321p1-p4, 3881, 3891, and B973 on 3,666 patients (1986 to 2001) with documented ages and follow-up data. Twenty-seven separate analyses, one for each different age cutoff (adjusting for MYCN and stage), tested age influence on outcome. The cutoff that maximized outcome difference between younger and older patients was selected. RESULTS Thirty-seven percent of patients were younger than 365 days, and 64% were > or = 365 days old (4-year event-free survival [EFS] rate +/- SE: 83% +/- 1% [n = 1,339] and 45% +/- 1% [n = 2,327], respectively; P < .0001). Graphical analyses revealed the continuous nature of the prognostic contribution of age to outcome. The optimal 460-day cutoff we selected maximized the outcome difference between younger and older patients. Forty-three percent were younger than 460 days, and 57% were > or = 460 days old (4-year EFS rate +/- SE: 82% +/- 1% [n = 1,589] and 42% +/- 1% [n = 2,077], respectively; P < .0001). Using a 460-day cutoff (assuming stage 4, MYCN-amplified patients remain high-risk), 5% of patients (365 to 460 days: 4-year EFS 92% +/- 3%; n = 135) fell into a lower risk group. CONCLUSION The prognostic contribution of age to outcome is continuous in nature. Within clinically relevant risk stratification, statistical support exists for an age cutoff of 460 days.

Chromosome 6p22 Locus Associated with Clinically Aggressive Neuroblastoma

BACKGROUND Neuroblastoma is a malignant condition of the developing sympathetic nervous system that most commonly affects young children and is often lethal. Its cause is not known. METHODS We performed a genomewide association study by first genotyping blood DNA samples from 1032 patients with neuroblastoma and 2043 control subjects of European descent using the Illumina HumanHap550 BeadChip. Samples from three independent groups of patients with neuroblastoma (a total of 720 patients) and 2128 control subjects were then genotyped to replicate significant associations. RESULTS We observed a significant association between neuroblastoma and the common minor alleles of three consecutive single-nucleotide polymorphisms (SNPs) at chromosome band 6p22 and containing the predicted genes FLJ22536 and FLJ44180 (P=1.71x10(-9) to 7.01x10(-10); allelic odds ratio, 1.39 to 1.40). Homozygosity for the at-risk G allele of the most significantly associated SNP, rs6939340, resulted in an increased likelihood of the development of neuroblastoma (odds ratio, 1.97; 95% confidence interval, 1.58 to 2.45). Subsequent genotyping of the three 6p22 SNPs in three independent case series confirmed our observation of an association (P=9.33x10(-15) at rs6939340 for joint analysis). Patients with neuroblastoma who were homozygous for the risk alleles at 6p22 were more likely to have metastatic (stage 4) disease (P=0.02), amplification of the MYCN oncogene in the tumor cells (P=0.006), and disease relapse (P=0.01). CONCLUSIONS A common genetic variation at chromosome band 6p22 is associated with susceptibility to neuroblastoma.

Antitumor Activity of Hu14.18-IL2 in Patients With Relapsed/Refractory Neuroblastoma: A Children's Oncology Group (COG) Phase II Study

PURPOSE The hu14.18-IL2 fusion protein consists of interleukin-2 molecularly linked to a humanized monoclonal antibody that recognizes the GD2 disialoganglioside expressed on neuroblastoma cells. This phase II study assessed the antitumor activity of hu14.18-IL2 in two strata of patients with recurrent or refractory neuroblastoma. PATIENTS AND METHODS Hu14.18-IL2 was given intravenously (12 mg/m(2)/daily) for 3 days every 4 weeks for patients with disease measurable by standard radiographic criteria (stratum 1) and for patients with disease evaluable only by [(123)I]metaiodobenzylguanidine (MIBG) scintigraphy and/or bone marrow (BM) histology (stratum 2). Response was established by independent radiology review as well as BM histology and immunocytology, and durability was assessed by repeat evaluation after more than 3 weeks. RESULTS Thirty-nine patients were enrolled (36 evaluable). No responses were seen in stratum 1 (n = 13). Of 23 evaluable patients in stratum 2, five patients (21.7%) responded; all had a complete response (CR) of 9, 13, 20, 30, and 35+ months duration. Grade 3 and 4 nonhematologic toxicities included capillary leak, hypoxia, pain, rash, allergic reaction, elevated transaminases, and hyperbilirubinemia. Two patients required dopamine for hypotension, and one patient required ventilatory support for hypoxia. Most toxicities were reversible within a few days of completing a treatment course and were expected based on phase I results. CONCLUSION Patients with disease evaluable only by MIBG and/or BM histology had a 21.7% CR rate to hu14.8-IL2, whereas patients with bulky disease did not respond. Hu14.18-IL2 warrants further testing in children with nonbulky high-risk neuroblastoma.