The quinoxaline antitumor agent (R+)XK469 mediates its effects by topoisomerase IIB inhibition. This report describes a 14 year-old with relapsed neuroblastoma who experienced disease stabilization for 14 months while receiving (R+)XK469 monotherapy. Due to this favorable response, laboratory studies were undertaken to determine efficacy in the preclinical setting. (R+)XK469 inhibited proliferation, caused G2 cell cycle arrest of neuroblastoma cells in vitro, and inhibited growth of neuroblastoma xenograft tumors. These preclinical results, coupled with the favorable clinical response, demonstrate that (R+)XK469 and similar anti-tumor agents may be effective in the treatment of high-risk neuroblastoma and warrant further testing.
neuroblastoma; topoisomerase inhibitor; relapse; phase I
Black patients with neuroblastoma have a higher prevalence of high-risk disease and worse outcome than white patients. We sought to investigate the relationship between genetic variation and the disparities in survival observed in neuroblastoma.
The analytic cohort was composed of 2709 patients. Principal components were used to assign patients to genomic ethnic clusters for survival analyses. Locus-specific ancestry was calculated for use in association analysis. The shorter spans of linkage disequilibrium in African populations may facilitate the fine mapping of causal variants in regions previously implicated by genome-wide association studies conducted primarily in patients of European descent. Thus, we evaluated 13 single nucleotide polymorphisms known to be associated with susceptibility to high-risk neuroblastoma from genome-wide association studies and all variants with highly divergent allele frequencies in reference African and European populations near the known susceptibility loci. All statistical tests were two-sided.
African genomic ancestry was associated with high-risk neuroblastoma (P = .007) and lower event-free survival (P = .04, hazard ratio = 1.4, 95% confidence interval = 1.05 to 1.80). rs1033069 within SPAG16 (sperm associated antigen 16) was determined to have higher risk allele frequency in the African reference population and statistically significant association with high-risk disease in patients of European and African ancestry (P = 6.42×10−5, false discovery rate < 0.0015) in the overall cohort. Multivariable analysis using an additive model demonstrated that the SPAG16 single nucleotide polymorphism contributes to the observed ethnic disparities in high-risk disease and survival.
Our study demonstrates that common genetic variation influences neuroblastoma phenotype and contributes to the ethnic disparities in survival observed and illustrates the value of trans-population mapping.
Epigenetic changes in pediatric neuroblastoma may contribute to the aggressive pathophysiology of this disease, but little is known about the basis for such changes. In this study, we examined a role for the DNA methyltransferase DNMT3B, in particular, the truncated isoform DNMT3B7 which is generated frequently in cancer. To investigate if aberrant DNMT3B transcripts alter DNA methylation, gene expression, and phenotypic character in neuroblastoma, we measured DNMT3B expression in primary tumors. Higher levels of DNMT3B7 were detected in differentiated ganglioneuroblastomas compared to undifferentiated neuroblastomas, suggesting that expression of DNMT3B7 may induce a less aggressive clinical phenotype. To test this hypothesis, we investigated the effects of enforced DNMT3B7 expression in neuroblastoma cells, finding a significant inhibition of cell proliferation in vitro and angiogenesis and tumor growth in vivo. DNMT3B7-positive cells had higher levels of total genomic methylation and a dramatic decrease in expression of the FOS and JUN family members that comprise AP1 transcription factors. Consistent with an established antagonistic relationship between AP1 expression and retinoic acid receptor activity, increased differentiation was seen in the DNMT3B7-expressing neuroblastoma cells following treatment with all-trans retinoic acid (ATRA) compared to controls. Our results indicate that DNMT3B7 modifies the epigenome in neuroblastoma cells to induce changes in gene expression, inhibit tumor growth, and increase sensitivity to ATRA.
DNMT3B; Neuroblastoma; DNA Methylation
The primary objective of Children's Oncology Group study P9641 was to demonstrate that surgery alone would achieve 3-year overall survival (OS) ≥ 95% for patients with asymptomatic International Neuroblastoma Staging System stages 2a and 2b neuroblastoma (NBL). Secondary objectives focused on other low-risk patients with NBL and on those who required chemotherapy according to protocol-defined criteria.
Patients and Methods
Patients underwent maximally safe resection of tumor. Chemotherapy was reserved for patients with, or at risk for, symptomatic disease, with less than 50% tumor resection at diagnosis, or with unresectable progressive disease after surgery alone.
For all 915 eligible patients, 5-year event-free survival (EFS) and OS were 89% ± 1% and 97% ± 1%, respectively. For patients with asymptomatic stage 2a or 2b disease, 5-year EFS and OS were 87% ± 2% and 96% ± 1%, respectively. Among patients with stage 2b disease, EFS and OS were significantly lower for those with unfavorable histology or diploid tumors, and OS was significantly lower for those ≥ 18 months old. For patients with stage 1 and 4s NBL, 5-year OS rates were 99% ± 1% and 91% ± 1%, respectively. Patients who required chemotherapy at diagnosis achieved 5-year OS of 98% ± 1%. Of all patients observed after surgery, 11.1% experienced recurrence or progression of disease.
Excellent survival rates can be achieved in asymptomatic low-risk patients with stages 2a and 2b NBL after surgery alone. Immediate use of chemotherapy may be restricted to a minority of patients with low-risk NBL. Patients with stage 2b disease who are older or have diploid or unfavorable histology tumors fare less well. Future studies will seek to refine risk classification.
Myeloablative chemoradiotherapy and immunomagnetically purged autologous bone marrow transplantation has been shown to improve outcome for patients with high-risk neuroblastoma. Currently, peripheral blood stem cells (PBSC) are infused after myeloablative therapy, but the effect of purging is unknown. We did a randomised study of tumour-selective PBSC purging in stem-cell transplantation for patients with high-risk neuroblastoma.
Between March 16, 2001, and Feb 24, 2006, children and young adults (<30 years) with high-risk neuroblastoma were randomly assigned at diagnosis by a web-based system (in a 1:1 ratio) to receive either nonpurged or immunomagnetically purged PBSC. Randomisation was done in blocks stratified by International Neuroblastoma Staging System stage, age, MYCN status, and International Neuroblastoma Pathology classification. Patients and treating physicians were not masked to treatment assignment. All patients were treated with six cycles of induction chemotherapy, myeloablative consolidation, and radiation therapy to the primary tumour site plus metaiodobenzylguanidine avid metastases present before myeloablative therapy, followed by oral isotretinoin. PBSC collection was done after two induction cycles. For purging, PBSC were mixed with carbonyl iron and phagocytic cells removed with samarium cobalt magnets. Remaining cells were mixed with immunomagnetic beads prepared with five monoclonal antibodies targeting neuroblastoma cell surface antigens and attached cells were removed using samarium cobalt magnets. Patients underwent autologous stem-cell transplantation with PBSC as randomly assigned after six cycles of induction therapy. The primary endpoint was event-free survival and was analysed by intention-to-treat. The trial is registered with ClinicalTrials.gov, number NCT00004188.
495 patients were enrolled, of whom 486 were randomly assigned to treatment: 243 patients to receive non-purged PBSC and 243 to received purged PBSC. PBSC were collected from 229 patients from the purged group and 236 patients from the non-purged group, and 180 patients from the purged group and 192 from the non-purged group received transplant. 5-year event-free survival was 40% (95% CI 33–46) in the purged group versus 36% (30–42) in the non-purged group (p=0·77); 5-year overall survival was 50% (95% CI 43–56) in the purged group compared with 51% (44–57) in the non-purged group (p=0·81). Toxic deaths occurred in 15 patients during induction (eight in the purged group and seven in the non-purged group) and 12 during consolidation (eight in the purged group and four in the non-purged group). The most common adverse event reported was grade 3 or worse stomatitis during both induction (87 of 242 patients in the purged group and 93 of 243 patients in the non-purged group) and consolidation (131 of 177 in the purged group vs 145 of 191 in the non-purged group). Serious adverse events during induction were grade 3 or higher decreased cardiac function (four of 242 in the purged group and five of 243 in the non-purged group) and elevated creatinine (five of 242 in the purged group and six of 243 non-purged group) and during consolidation were sinusoidal obstructive syndrome (12 of 177 in the purged group and 17 of 191 in the non-purged group), acute vascular leak (11 of 177 in the purged group and nine of 191 in the non-purged group), and decreased cardiac function (one of 177 in the purged group and four of 191 in the non-purged group).
Immunomagnetic purging of PBSC for autologous stem-cell transplantation did not improve outcome, perhaps because of incomplete purging or residual tumour in patients. Non-purged PBSC are acceptable for support of myeloablative therapy of high-risk neuroblastoma.
National Cancer Institute and Alex’s Lemonade Stand Foundation.
Neuroblastoma is the most common extracranial solid tumor of childhood, and the outcomes for children with high-risk and relapsed disease remain poor. However, new international strategies for risk stratification and for treatment based on novel tumor targets and including immunotherapy are being employed in attempts to improve the outcomes of children with neuroblastoma. A new international neuroblastoma risk classification system has been developed which is being incorporated into cooperative group clinical trials in North America, Japan, and Europe, resulting in standardized approaches for the initial evaluation and treatment stratification of neuroblastoma patients. Furthermore, novel treatment regimens are being developed based on improved understanding of neuroblastoma biology and on the recruitment of the immune system to specifically target neuroblastoma tumors. These approaches will lead to new therapeutic strategies that likely will improve the outcomes for children with neuroblastoma worldwide.
immunotherapy; INRG; neuroblastoma
To assess the feasibility of adding dose-intensive topotecan and cyclophosphamide to induction therapy for newly diagnosed high-risk neuroblastoma (HRNB).
Patients and Methods
Enrolled patients received two cycles of topotecan (approximately 1.2 mg/m2/d) and cyclophosphamide (400 mg/m2/d) for 5 days followed by four cycles of multiagent chemotherapy (Memorial Sloan-Kettering Cancer Center [MSKCC] regimen). Pharmacokinetically guided topotecan dosing (target systemic exposure with area under the curve of 50 to 70 ng/mL/hr) was performed. Peripheral-blood stem cell (PBSC) harvest and surgical resection of residual primary tumor occurred after cycles 2 and 5, respectively. Patients achieving at least a partial response received myeloablative chemotherapy with PBSC rescue and radiation to the presurgical primary tumor volume. Oral 13-cis-retinoic acid maintenance therapy was administered twice daily for 14 days in six 28-day cycles.
Thirty-one patients were enrolled onto the study. No deaths related to toxicity or dose-limiting toxicities occurred during induction. Mucositis rarely occurred after topotecan cycles (9.7%) in contrast to 30% after MSKCC cycles. Thirty patients underwent PBSC collection with median 31.1 × 106 CD34+ cells/kg (range, 1.8 to 541.8 × 106 CD34+ cells/kg), all negative for tumor contamination by immunocytochemical analysis. Targeted topotecan systemic exposure was achieved in 26 (84%) of 31 patients. At the end of induction, 26 patients (84%) had tumor response and one patient had progressive disease. In the overall cohort, 3-year event-free and overall survival were 37.8% ± 9.4% and 57.1% ± 9.4%, respectively.
This pilot induction regimen was well tolerated with expected and reversible toxicities. These data support investigation of efficacy in a phase III clinical trial for newly diagnosed HRNB.
Patients with neuroblastoma younger than 12 months of age with a 4S pattern of disease (metastases limited to liver, skin, bone marrow) have better outcomes than infants with stage 4 disease. The new International Neuroblastoma Risk Group (INRG) staging system extends age to 18 months for the 4S pattern. Our aim was to determine which prognostic features could be used for optimal risk classification among patients younger than 18 months with metastatic disease.
Event-free survival (EFS) and overall survival were analyzed by log-rank tests, Cox models, and survival tree regression for 656 infants with stage 4S neuroblastoma younger than 12 months of age and 1,019 patients with stage 4 disease younger than 18 months of age in the INRG database.
Unfavorable biologic features were more frequent in infants with stage 4 disease than in infants with 4S tumors and higher overall in those age 12 to 18 months (although not different for stage 4 v 4S pattern). EFS was significantly better for infants younger than 12 months with 4S pattern than with stage 4 disease (P < .01) but similar for toddlers age 12 to 18 months with stage 4 versus 4S pattern. Among 717 patients with stage 4S pattern, patients age 12 to 18 months had worse EFS than those age younger than 12 months (P < .01). MYCN, 11q, mitosis-karyorrhexis index (MKI), ploidy, and lactate dehydrogenase were independently statistically significant predictors of EFS and more highly predictive than age or metastatic pattern. MYCN, 11q, MKI, histology, and 1p were combined in a survival tree for improved risk stratification.
Tumor biology is more critical than age or metastatic pattern for prognosis of patients age younger than 18 months with metastatic neuroblastoma and should be considered for risk stratification.
Survival after neuroblastoma relapse is poor. Understanding the relationship between clinical and biologic features and outcome after relapse may help in selection of optimal therapy. Our aim was to determine which factors were significantly predictive of postrelapse overall survival (OS) in patients with recurrent neuroblastoma—particularly whether time from diagnosis to first relapse (TTFR) was a significant predictor of OS.
Patients and Methods
Patients with first relapse/progression were identified in the International Neuroblastoma Risk Group (INRG) database. Time from study enrollment until first event and OS time starting from first event were calculated. Cox regression models were used to calculate the hazard ratio of increased death risk and perform survival tree regression. TTFR was tested in a multivariable Cox model with other factors.
In the INRG database (N = 8,800), 2,266 patients experienced first progression/relapse. Median time to relapse was 13.2 months (range, 1 day to 11.4 years). Five-year OS from time of first event was 20% (SE, ± 1%). TTFR was statistically significantly associated with OS time in a nonlinear relationship; patients with TTFR of 36 months or longer had the lowest risk of death, followed by patients who relapsed in the period of 0 to less than 6 months or 18 to 36 months. Patients who relapsed between 6 and 18 months after diagnosis had the highest risk of death. TTFR, age, International Neuroblastoma Staging System stage, and MYCN copy number status were independently predictive of postrelapse OS in multivariable analysis.
Age, stage, MYCN status, and TTFR are significant prognostic factors for postrelapse survival and may help in the design of clinical trials evaluating novel agents.
Stromal cells play a central role in the regulation of tumor angiogenesis. Recent studies have shown that stromal myofibroblasts [cancer-associated fibroblasts] actively promote tumor growth and enhance tumor angiogenesis in many types of adult carcinomas. To evaluate the role cancer-associated fibroblasts play in neuroblastoma angiogenesis and investigate their relationship to stromal Schwann cells, we quantified cancer-associated fibroblasts in 60 primary neuroblastoma tumors and in a novel neuroblastoma xenograft model in which murine Schwann cells were induced to infiltrate into the tumor stroma. Tumor sections were examined for presence of microvascular proliferation, a hallmark of tumor angiogenesis. Cancer-associated fibroblasts were characterized by positive immunostaining for α-smooth muscle actin (α-SMA) and were distinguished from pericytes by staining negatively for high-molecular weight caldesmon. α-SMA positive cells were quantified and their number was defined as high when >1.0% of the area was positive. Associations between high cancer-associated fibroblast number, microvascular proliferation, and established prognosticators were analyzed. High numbers of cancer-associated fibroblasts were associated with Schwannian Stroma-poor histopathology and microvascular proliferation. Thirty-seven (80%) of the 46 Schwannian Stroma-poor tumors had high numbers of cancer-associated fibroblasts in the tumor stroma compared to only 2 (14%) of the 14 Schwannian Stroma-rich/dominant tumors (p<0.001). Thirty-three (89%) of 37 tumors with microvascular proliferation had high numbers of cancer-associated fibroblasts compared to 9 (40%) of 22 tumors without microvascular proliferation (p <0.001). In the xenografts with infiltrating Schwann cells (n=10), the number of cancer-associated fibroblasts/mm2 was ~7-fold less than in the control xenografts without stromal Schwann cells (n=9) (mean of 51 ± 30 vs 368 ± 105, respectively; p<0.001). Thus, cancer-associated fibroblasts were inversely associated with presence of Schwann cells, suggesting that Schwann cells may prevent the activation of fibroblasts. A deeper understanding of the role cancer-associated fibroblasts play in neuroblastoma angiogenesis may guide future development of stroma-directed therapeutic strategies.
Cancer-associated fibroblast; Neuroblastoma; Schwannian Stroma
Although health disparities are well-described for many cancers, little is known about racial and ethnic disparities in neuroblastoma. To evaluate differences in disease presentation and survival by race and ethnicity, data from the Children's Oncology Group (COG) were analyzed.
Patients and Methods
The racial/ethnic differences in clinical and biologic risk factors, and outcome of patients with neuroblastoma enrolled on COG ANBL00B1 between 2001 and 2009 were investigated.
A total of 3,539 patients (white, 72%; black, 12%; Hispanic, 12%; Asian, 4%; and Native American, < 1%) with neuroblastoma were included. The 5-year event-free survival (EFS) rates were 67% for whites (95% CI, 65% to 69%), 69% for Hispanics (95% CI, 63% to 74%), 62% for Asians (95% CI, 51% to 71%), 56% for blacks (95% CI, 50% to 62%), and 37% for Native American (95% CI, 17% to 58%). Blacks (P < .001) and Native Americans (P = .04) had a higher prevalence of high-risk disease than whites, and significantly worse EFS (P = .01 and P = .002, respectively). Adjustment for risk group abrogated these differences. However, closer examination of the EFS among high-risk patients who remained event free for 2 years or longer, revealed a higher prevalence of late-occurring events among blacks compared with whites (hazard ratio, 1.5; 95% CI, 1.0 to 2.3; P = .04).
Black and Native American patients with neuroblastoma have a higher prevalence of high-risk disease, accounting for their worse EFS when compared with whites. The higher prevalence of late-occurring events among blacks with high-risk disease suggests that this population may be more resistant to chemotherapy. Studies focused on delineating the genetic basis for the racial disparities observed in this study are planned.
Of 4,706 peripheral neuroblastic tumors (pNTs) registered on the Children’s Cancer Group and Children’s Oncology Group Neuroblastoma Study between 1989 and 2010, 51 cases (1.1%) had genotype-phenotype discordance characterized by MYCN amplification (indicating poor prognosis) and Favorable Histology (indicating better prognosis).
To distinguish prognostic subgroups in the genotype-phenotype discordant pNTs, two subgroups, “conventional” and “bull’s eye”, were identified based on the nuclear morphology. The “conventional” tumors (35 cases) included: Neuroblastoma, Poorly differentiated subtype (NB-PD, 26 cases) with “salt-and-pepper” nuclei; Neuroblastoma, Differentiating subtype (4 cases); Ganglioneuroblastoma, Intermixed (3 cases); and Ganglioneuroma, Maturing subtype (2 cases). The “bull’s eye” tumors included NB-PD with prominent nucleoli (16 cases). Clinicopathologic characteristics of these two subgroups were analyzed. N-myc protein expression was tested immunohistochemically on available tumors.
No significant difference was found between these two subgroups in the distribution of prognostic factors such as age at diagnosis, clinical stage, histopathology category/subtype, mitosis-karyorrhexis index, ploidy, 1p LOH, and unbalanced 11qLOH. However, prognosis of the patients with “conventional” tumors (5-year EFS 85.7±12.2%; OS 89.3±10.3%) was significantly better than those with “bull’s eye” tumors (EFS 31.3±13.0%; OS 42.9±16.2%) (P=0.0010 and 0.0008, respectively). Immunohistochemically all (11/11) tested “conventional” tumors were negative, and 10/11 tested “bull’s eye” tumors were positive for N-myc protein expression.
Based on the presence or absence of prominent nucleoli (the putative site of RNA synthesis/accumulation leading to N-myc protein expression), two prognostic subgroups, “conventional” with a better prognosis and “bull’s eye” with a poor prognosis, were distinguished among the genotype-phenotype discordant pNTs.
neuroblastoma; International Neuroblastoma Pathology Classification; MYCN; genotype-phenotype correlation; prognosis; immunohistochemistry
Increasing treatment intensity has improved outcomes for children with neuroblastoma. We performed a pilot study in the Children’s Oncology Group (COG) to assess feasibility and toxicity of a tandem myeloablative regimen without total body irradiation (TBI) supported by autologous CD34 selected peripheral blood stem cells. Forty-one patients with high-risk neuroblastoma were enrolled; eight patients did not receive any myeloablative consolidation procedure, and seven received only one. Two patients out of 41 (4.9%) experienced transplant-related mortality. CD34 selection was discontinued after subjects were enrolled due to serious viral illness. From the time of study enrollment, the overall 3-year event-free survival (EFS) and overall survival (OS) were 44.8±9.6% and 59.2±9.2% (N=41). These results demonstrate that tandem transplantation in the cooperative group setting is feasible and support a randomized comparison of single versus tandem myeloablative consolidation with PBSC support for high-risk neuroblastoma.
pediatric; neuroblastoma; tandem transplant; hematopoietic stem cell transplant
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 (Children's 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.
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 ≤ 85%, ≥ 50% to ≤ 75%, or less than 50% were classified as very low risk, low risk, intermediate risk, or high risk, respectively.
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.
The amazing successes in cure rates for children with cancer over the last century have come in large part from identifying clinical, genetic and molecular variables associated with response to therapy in large cooperative clinical trials and stratifying therapies according to the predicted risk of relapse. There is an expanding interest in identifying germline genomic variants, as opposed to genetic variants within the tumor, that are associated with susceptibility to toxicity and for risk of relapse. This review highlights the most important germline pharmacogenetic and pharmacogenomic studies in pediatric oncology. Incorporation of germline genomics into risk-adapted therapies will likely lead to safer and more effective treatments for children with cancer.
Pediatric Oncology; Pharmacogenetics; Pharmacogenomics; Genome-wide Association Studies; Single Nucleotide Polymorphisms
Clinical information, “-omic” datasets, and tissue samples are difficult to harmonize and manage for data mining. We have developed a platform for storing clinical research data while providing access to associated data from other information stores. Data on 34 metrics from 11,000 neuroblastoma patients were instantiated into a database. The Django web framework was used to create a model for rapid development of tools and views with a front-end interface for generating complex queries. Working with Nationwide Children’s Hospital, we can now consume their tissue inventory data through an API. The end-user sees the number of patients who both match their search and have tissue available. Since initial implementation, the current tasks revolve around developing a governance structure and the necessary data use agreements. Efforts now are to (1) update the data with 5000 more patients, and (2) link to genomic data stores, facilitating disparate data acquisition for research studies.
To determine the response rate to oral capsular fenretinide in children with recurrent or biopsy proven refractory high-risk neuroblastoma.
Patients received 7 days of fenretinide: 2475 mg/m2/day divided TID (<18 years) or 1800 mg/m2/day divided BID (≥18 years) every 21 days for a maximum of 30 courses. Patients with stable or responding disease after course 30 could request additional compassionate courses. Best response by course 8 was evaluated in Stratum 1 (measurable disease on CT/MRI +/− bone marrow and/or MIBG avid sites) and Stratum 2 (bone marrow and/or MIBG avid sites only).
Sixty-two eligible patients, median age 5 years (range 0.6–19.9), were treated in Stratum 1 (n=38) and Stratum 2 (n=24). One partial response (PR) was seen in Stratum 2 (n=24 evaluable). No responses were seen in Stratum 1 (n=35 evaluable). Prolonged stable disease (SD) was seen in 7 patients in Stratum 1 and 6 patients in Stratum 2 for 4–45+ (median 15) courses. Median time to progression was 40 days (range 17–506) for Stratum 1 and 48 days (range 17–892) for Stratum 2. Mean 4-HPR steady state trough plasma concentrations were 7.25 µM (coefficient of variation 40–56%) at day 7 course 1. Toxicities were mild and reversible.
Although neither stratum met protocol criteria for efficacy, 1 PR + 13 prolonged SD occurred in 14/59 (24%) of evaluable patients. Low bioavailability may have limited fenretinide activity. Novel fenretinide formulations with improved bioavailability are currently in pediatric Phase I studies.
fenretinide; neuroblastoma; Phase II; ANBL0321
This phase II study was conducted to determine the response rate associated with use of irinotecan and temozolomide for children with relapsed/refractory neuroblastoma.
Patients and Methods
Patients with relapsed/refractory neuroblastoma measurable by cross-sectional imaging (stratum 1) or assessable by bone marrow aspirate/biopsy or metaiodobenzylguanidine (MIBG) scan (stratum 2) received irinotecan (10 mg/m2/dose 5 days a week for 2 weeks) and temozolomide (100 mg/m2/dose for 5 days) every 3 weeks. Response was assessed after three and six courses using International Neuroblastoma Response Criteria. Of the first 25 evaluable patients on a given stratum, five or more patients with complete or partial responses were required to conclude that further study would be merited.
Fifty-five eligible patients were enrolled. The objective response rate was 15%. Fourteen patients (50%) on stratum 1 and 15 patients (56%) on stratum 2 had stable disease. Objective responses were observed in three of the first 25 evaluable patients on stratum 1 and five of the first 25 evaluable patients on stratum 2. Less than 6% of patients experienced ≥ grade 3 diarrhea. Although neutropenia was observed, less than 10% of patients developed evidence of infection while neutropenic.
The combination of irinotecan and temozolomide was well tolerated. The objective response rate of 19% in stratum 2 suggests that this combination may be effective for patients with neuroblastoma detectable by MIBG or marrow analysis. Although fewer objective responses were observed in patients with disease measurable by computed tomography/magnetic resonance imaging, patients in both strata seem to have derived clinical benefit from this therapy.
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/m2/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 [123I]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.
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.
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.
Secreted Protein Acidic and Rich in Cysteine (SPARC) is one of the major non-structural proteins of the extracellular matrix (ECM) in remodeling tissues. The functional significance of SPARC is emphasized by its origin in the first multicellular organisms and its high degree of evolutionary conservation. Although SPARC has been shown to act as a critical modulator of ECM remodeling with profound effects on tissue physiology and architecture, no plausible molecular mechanism of its action has been proposed. In the present study, we demonstrate that SPARC mediates the disassembly and degradation of ECM networks by functioning as a matricellular chaperone. While it has low affinity to its targets inside the cells where the Ca2+ concentrations are low, high extracellular concentrations of Ca2+ activate binding to multiple ECM proteins, including collagens. We demonstrated that in vitro, this leads to the inhibition of collagen I fibrillogenesis and disassembly of pre-formed collagen I fibrils by SPARC at high Ca2+ concentrations. In cell culture, exogenous SPARC was internalized by the fibroblast cells in a time- and concentration-dependent manner. Pulse-chase assay further revealed that internalized SPARC is quickly released outside the cell, demonstrating that SPARC shuttles between the cell and ECM. Fluorescently labeled collagen I, fibronectin, vitronectin, and laminin were co-internalized with SPARC by fibroblasts, and semi-quantitative Western blot showed that SPARC mediates internalization of collagen I. Using a novel 3-dimentional model of fluorescent ECM networks pre-deposited by live fibroblasts, we demonstrated that degradation of ECM depends on the chaperone activity of SPARC. These results indicate that SPARC may represent a new class of scavenger chaperones, which mediate ECM degradation, remodeling and repair by disassembling ECM networks and shuttling ECM proteins into the cell. Further understanding of this mechanism may provide insight into the pathogenesis of matrix-associated disorders and lead to the novel treatment strategies.
Single-agent topotecan (TOPO) and combination topotecan and cyclophosphamide (TOPO/CTX) were compared in a phase II randomized trial in relapsed/refractory neuroblastoma. Because responders often underwent further therapies, novel statistical methods were required to compare the long-term outcome of the two treatments.
Patients and Methods
Children with refractory/recurrent neuroblastoma (only one prior aggressive chemotherapy regimen) were randomly assigned to daily 5-day topotecan (2 mg/m2) or combination topotecan (0.75 mg/m2) and cyclophosphamide (250 mg/m2). A randomized two-stage group sequential design enrolled 119 eligible patients. Toxicity and response were estimated. Long-term outcome of protocol therapy was assessed using novel methods—causal inference—which allowed adjustment for the confounding effect of off-study therapies.
Seven more responses were observed for TOPO/CTX (complete response [CR] plus partial response [PR], 18 [32%] of 57) than TOPO (CR+PR, 11 [19%] of 59;P = .081); toxicity was similar. At 3 years, progression-free survival (PFS) and overall survival (OS) were 4% ± 2% and 15% ± 4%, respectively. PFS was significantly better for TOPO/CTX (P = .029); there was no difference in OS. Older age at diagnosis and lack of MYCN amplification predicted increased OS (P < .05). Adjusting for randomized treatment effect and subsequent autologous stem-cell transplantation, there was no difference between TOPO and TOPO/CTX in terms of the proportion alive at 2 years.
TOPO/CTX was superior to TOPO in terms of PFS, but there was no OS difference. After adjustment for subsequent therapies, no difference was detected in the proportion alive at 2 years. Causal inference methods for assessing long-term outcomes of phase II therapies after subsequent treatment can elucidate effects of initial therapies.
Although the prognostic value of the ATP-binding cassette, subfamily C (ABCC) transporters in childhood neuroblastoma is usually attributed to their role in cytotoxic drug efflux, certain observations have suggested that these multidrug transporters might contribute to the malignant phenotype independent of cytotoxic drug efflux.
A v-myc myelocytomatosis viral related oncogene, neuroblastoma derived (MYCN)–driven transgenic mouse neuroblastoma model was crossed with an Abcc1-deficient mouse strain (658 hMYCN1/−, 205 hMYCN+/1 mice) or, alternatively, treated with the ABCC1 inhibitor, Reversan (n = 20). ABCC genes were suppressed using short interfering RNA or overexpressed by stable transfection in neuroblastoma cell lines BE(2)-C, SH-EP, and SH-SY5Y, which were then assessed for wound closure ability, clonogenic capacity, morphological differentiation, and cell growth. Real-time quantitative polymerase chain reaction was used to examine the clinical significance of ABCC family gene expression in a large prospectively accrued cohort of patients (n = 209) with primary neuroblastomas. Kaplan–Meier survival analysis and Cox regression were used to test for associations with event-free and overall survival. Except where noted, all statistical tests were two-sided.
Inhibition of ABCC1 statistically significantly inhibited neuroblastoma development in hMYCN transgenic mice (mean age for palpable tumor: treated mice, 47.2 days; control mice, 41.9 days; hazard ratio [HR] = 9.3, 95% confidence interval [CI] = 2.65 to 32; P < .001). Suppression of ABCC1 in vitro inhibited wound closure (P < .001) and clonogenicity (P = .006); suppression of ABCC4 enhanced morphological differentiation (P < .001) and inhibited cell growth (P < .001). Analysis of 209 neuroblastoma patient tumors revealed that, in contrast with ABCC1 and ABCC4, low rather than high ABCC3 expression was associated with reduced event-free survival (HR of recurrence or death = 2.4, 95% CI = 1.4 to 4.2; P = .001), with 23 of 53 patients with low ABCC3 expression experiencing recurrence or death compared with 31 of 155 patients with high ABCC3. Moreover, overexpression of ABCC3 in vitro inhibited neuroblastoma cell migration (P < .001) and clonogenicity (P = .03). The combined expression of ABCC1, ABCC3, and ABCC4 was associated with patients having an adverse event, such that of the 12 patients with the “poor prognosis” expression pattern, 10 experienced recurrence or death (HR of recurrence or death = 12.3, 95% CI = 6 to 27; P < .001).
ABCC transporters can affect neuroblastoma biology independently of their role in chemotherapeutic drug efflux, enhancing their potential as targets for therapeutic intervention.
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.
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.
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).
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.
The survival rate among patients with intermediate-risk neuroblastoma who receive dose-intensive chemotherapy is excellent, but the survival rate among patients who receive reduced doses of chemotherapy for shorter periods of time is not known.
We conducted a prospective, phase 3, nonrandomized trial to determine whether a 3-year estimated overall survival of more than 90% could be maintained with reductions in the duration of therapy and drug doses, using a tumor biology-based therapy assignment. Eligible patients had newly diagnosed, intermediate-risk neuroblastoma without MYCN amplification; these patients included infants (<365 days of age) who had stage 3 or 4 disease, children (≥365 days of age) who had stage 3 tumors with favorable histopathological features, and infants who had stage 4S disease with a diploid DNA index or unfavorable histopathological features. Patients who had disease with favorable histopathological features and hyperdiploidy were assigned to four cycles of chemotherapy, and those with an incomplete response or either unfavorable feature were assigned to eight cycles.
Between 1997 and 2005, a total of 479 eligible patients were enrolled in this trial (270 patients with stage 3 disease, 178 with stage 4 disease, and 31 with stage 4S disease). A total of 323 patients had tumors with favorable biologic features, and 141 had tumors with unfavorable biologic features. Ploidy, but not histopathological features, was significantly predictive of the outcome. Severe adverse events without disease progression occurred in 10 patients (2.1%), including secondary leukemia (in 3 patients), death from infection (in 3 patients), and death at surgery (in 4 patients). The 3-year estimate (±SE) of overall survival for the entire group was 96±1%, with an overall survival rate of 98±1% among patients who had tumors with favorable biologic features and 93±2% among patients who had tumors with unfavorable biologic features.
A very high rate of survival among patients with intermediate-risk neuroblastoma was achieved with a biologically based treatment assignment involving a substantially reduced duration of chemotherapy and reduced doses of chemotherapeutic agents as compared with the regimens used in earlier trials. These data provide support for further reduction in chemotherapy with more refined risk stratification. (Funded by the National Cancer Institute; ClinicalTrials.gov number, NCT00003093.)
ABT-751, an orally bioavailable sulfonamide, binds β-tubulin to inhibit microtubule polymerization. To describe response and event free survival (EFS) in children with neuroblastoma and other solid tumors receiving ABT-751, assess in vitro cytotoxicity of ABT-751 and evaluate the effect of ABT-751 on tubulin polymerization in peripheral blood mononuclear cells (PBMC) and pediatric tumor cell lines.
Patients with neuroblastoma (n=50) or other solid tumors (n=26) enrolled on the ABT-751 pediatric phase I and pilot trials were reviewed. The sulforhodamine B (SRB) and ACEA Real-Time Cell Electronic Sensing (RT-CES) assays were used to determine the in vitro cytotoxicity. Pharmacodynamic effects on tubulin polymerization/depolymerization were assessed by western blot and confocal microscopy using antibodies specific for post-translational modifications of polymerized tubulin.
Forty-five patients with neuroblastoma were evaluated for anti-tumor response. No complete or partial responses were documented. The median EFS was 9.3 weeks for children with neuroblastoma and 3.3 weeks for children other solid tumors (P<0.0001). The ABT-751 IC50 was 0.6–2.6 mcM in neuroblastoma and 0.7–4.6 mcM in other solid tumor cell lines. Following drug exposure, polymerized tubulin decreased in a concentration and time dependent manner in cell lines.
In children treated with ABT-751, the EFS is longer in children with neuroblastoma as compared to other diagnoses. In vitro, ABT-751 was cytotoxic at concentrations tolerable in children. Effects of ABT-751 on polymerization and microtubule structure were time and dose-dependent but not dependent on tumor type.
neuroblastoma; ABT-751; cytotoxicity; pediatric; tubulin