Tomoko Iehara

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.

Circulating muscle-specific microRNA, miR-206, as a potential diagnostic marker for rhabdomyosarcoma

Presently there is no serum biomarker of rhabdomyosarcoma (RMS). Several studies have shown that profiles of microRNA (miRNA) expression differ among tumor types. Here we evaluated the feasibility of using muscle-specific miRNAs (miR-1, -133a, -133b and -206) as biomarkers of RMS. Expression of muscle-specific miRNAs, especially miR-206, was significantly higher in RMS cell lines than in other tumor cell lines, as well as in RMS tumor specimens. Further, serum levels of muscle-specific miRNAs were significantly higher in patients with RMS tumors than in patients with non-RMS tumors. Normalized serum miR-206 expression level could be used to differentiate between RMS and non-RMS tumors, with sensitivity of 1.0 and specificity of 0.913. These results raise the possibility of using circulating muscle-specific miRNAs, especially miR-206, as landmark biomarkers for RMS.

Clinical and Biologic Features Predictive of Survival After Relapse of Neuroblastoma: A Report From the International Neuroblastoma Risk Group Project

PURPOSE 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. RESULTS 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. CONCLUSION 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.

Restoration of p53 Pathway by Nutlin-3 Induces Cell Cycle Arrest and Apoptosis in Human Rhabdomyosarcoma Cells

Purpose: Seventy to eighty percent of rhabdomyosarcoma (RMS) tumors retain wild-type p53. The tumor suppressor p53 plays a central role in inducing cell cycle arrest or apoptosis in response to various stresses. p53 protein levels are regulated by MDM2 through ubiquitin-dependent degradation. In this study, we evaluated whether nutlin-3, a recently developed small-molecule antagonist of MDM2, has an effect on p53-dependent cell cycle arrest and apoptosis in cultured human RMS cell lines. Experimental Design: Five RMS cell lines with different p53 statuses and MDM2 expression levels were treated with nutlin-3. Gene expression patterns, cell viability, cell cycle, and apoptosis after nutlin-3 treatment, and antitumor activity of combination treatment with vincristine or actinomycin D were assessed. Results: Significant p53 activation was observed in wild-type p53 cell lines after nutlin-3 treatment. p53 activation led to cell cycle arrest in parallel with increased p21 expression. Furthermore, these cell lines underwent p53-dependent apoptosis, concomitant with elevation of proapoptotic genes and activation of caspase-3. The effect of nutlin-3 was almost the same in terms of half maximal inhibitory concentration and apoptosis whether or not MDM2 was overexpressed. Nutlin-3 did not induce either cell cycle arrest or apoptosis in p53 mutant cell lines. A combination of vincristine or actinomycin D with nutlin-3 enhanced the antitumor activity in RMS cell lines with wild-type p53. Conclusions: Nutlin-3 effectively restored p53 function in both normal MDM2 expression and MDM2 overexpression RMS cell lines with wild-type p53. p53 restoration therapy is a potential therapeutic strategy for refractory RMS with wild-type p53.

Fenretinide induces sustained‐activation of JNK/p38 MAPK and apoptosis in a reactive oxygen species‐dependent manner in neuroblastoma cells

Fenretinide, which mediates apoptosis in neuroblastoma cells, is being considered as a novel therapeutic for neuroblastoma. The cytotoxic mechanisms of fenretinide, however, have not been fully elucidated. Sustained‐activation of JNK and p38 MAPK signaling has been shown recently to have a pivotal role in stress‐induced apoptosis. Whether fenretinide activates the signaling in neuroblastoma cells is not known. In the present study, fenretinide induced sustained‐activation of both JNK and p38 MAPK in neuroblastoma cells. Pretreatment with the antioxidant L‐ascorbic acid almost completely inhibited the accumulation of fenretinide‐induced intracellular reactive oxygen species (ROS), activation of JNK and p38 MAPK and apoptosis. Intracellular ROS production and activation of stress signaling was not altered by fenretinide in resistant neuroblastoma cells. Our study demonstrates that in neuroblastoma cells, fenretinide induces sustained‐activation of JNK and p38 MAPK in an ROS‐dependent manner and indicates that JNK and p38 MAPK signaling might mediate fenretinide‐induced apoptosis. Our results also indicate that suppression of the fenretinide‐induced ROS productive system and the downstream JNK and p38 MAPK signaling pathways causes neuroblastoma cells to become resistant to fenretinide.

Effectiveness of screening for neuroblastoma at 6 months of age: a retrospective population-based cohort study

BACKGROUND In Japan, a nationwide programme between 1984 and 2003 screened all infants for urinary catecholamine metabolites as a marker for neuroblastoma. Before 1989, this was done by qualitative spot tests for vanillylmandelic acid in urine, and subsequently by quantitative assay with high-performance liquid chromatography (HPLC). However, the Japanese government stopped the mass-screening programme in 2003, after reports that it did not reduce mortality due to neuroblastoma. We aimed to assess the effectiveness of the programme, by comparing the rates of incidence and mortality from neuroblastomas diagnosed before 6 years of age in three cohorts. METHODS We did a retrospective population-based cohort study on all children born between 1980 and 1998, except for a 2-year period from 1984. We divided these 22,289,695 children into three cohorts: children born before screening in 1980-83 (n=6,130,423); those born during qualitative screening in 1986-89 (n=5,290,412); and those born during quantitative screening 1990-98 (n=10,868,860). We used databases from hospitals, screening centres, and national cancer registries. Cases of neuroblastoma were followed up for a mean of 78.7 months. FINDINGS 21.56 cases of neuroblastoma per 100,000 births over 72 months were identified in the qualitatively screened group (relative risk [RR] 1.87, 95% CI 1.66-2.10), and 29.80 cases per 100,000 births over 72 months in the quantitatively screened group (RR 2.58, 2.33-2.86). The cumulative incidence of neuroblastoma in the prescreening cohort (11.56 cases per 100,000 births over 72 months) was lower than that in other cohorts (p<0.0001 for all comparisons), but more neuroblastomas were diagnosed after 24 months of age in this cohort (p=0.0002 for qualitative screening vs prescreening, p<0.0001 for quantitative screening vs prescreening). Cumulative mortality was lower in the qualitative screening (3.90 cases per 100,000 livebirths over 72 months) and quantitative screening cohorts (2.83 cases) than in the prescreening cohort (5.38 cases). Compared with the prescreening cohort, the relative risk of mortality was 0.73 (95% CI 0.58-0.90) for qualitative screening, and 0.53 (0.42-0.63) for quantitative screening. Mortality rates for both the qualitative and quantitative screening groups were lower than were those for the prescreening cohort (p=0.0041 for prescreening vs qualitative screening, p<0.0001 for prescreening vs quantitative screening). INTERPRETATION More infantile neuroblastomas were recorded in children who were screened for neuroblastoma at 6 months of age than in those who were not. The mortality rate from neuroblastoma in children who were screened at 6 months was lower than that in the prescreening cohort, especially in children screened by quantitative HPLC. Any new screening programme should aim to decrease mortality, but also to minimise overdiagnosis of tumours with favourable prognoses (eg, by screening children at 18 months).

Prediction of MYCN Amplification in Neuroblastoma Using Serum DNA and Real-Time Quantitative Polymerase Chain Reaction

PURPOSE MYCN amplification (MNA) indicates a poor prognosis in neuroblastoma (NB) and is routinely assayed for therapy stratification. We aimed to develop a diagnostic tool to predict MYCN status using serum DNA, which, in cancer patients, predominantly originates from tumor-released DNA. PATIENTS AND METHODS Using DNA-based real-time quantitative polymerase chain reaction, we simultaneously quantified MYCN (2p24) and a reference gene, NAGK (2p12), and evaluated MYCN copy number as an MYCN/NAGK (M/N) ratio in 87 NB patients whose MYCN status had been determined by Southern blotting. Of these patients, 17 had MYCN-amplified NB, and 70 had nonamplified NB. RESULTS The serum M/N ratio in the MNA group (median, 199.32; range, 17.1 to 901.6; 99% CI, 107.0 to 528.7) was significantly (P < .001) higher than the ratio in the non-MNA group (median, 0.87; range, 0.25 to 4.6; 99% CI, 0.82 to 1.26; Mann-Whitney U test). The sensitivity and specificity of the serum M/N ratio as a diagnostic test were both 100% when the serum M/N ratio cutoff was set at 10.0. Among six MNA patients whose clinical courses were followed, the serum ratios decreased to the normal range in the patients in remission (n = 3), whereas the ratios increased to high levels in the patients who relapsed (n = 2) or failed to achieve remission (n = 1). CONCLUSION Measurement of the serum M/N ratio seems to be a promising method for accurately assessing MYCN status in NB, although a larger set of patients needs to be examined to confirm this result.

Changes over three decades in outcome and the prognostic influence of age-at-diagnosis in young patients with neuroblastoma: A report from the International Neuroblastoma Risk Group Project

PURPOSE Increasing age has been an adverse risk factor in children with neuroblastoma (NB) since the 1970s, with a 12-month age-at-diagnosis cut-off for treatment stratification. Over the last 30 years, treatment intensity for children >12 months with advanced-stage disease has increased; to investigate if this strategy has improved outcome and/or reduced the prognostic influence of age, we analysed the International Neuroblastoma Risk Group (INRG) database. PATIENTS AND METHODS Data from 11,037 children with NB (1974-2002) from Australia, Europe, Japan, North America. Cox modelling of event-free survival (EFS) tested if the era and prognostic significance of age-of-diagnosis, adjusted for bone marrow (BM) metastases and MYCN status, effects on outcome had changed. RESULTS Outcome improved over time: 3-year EFS 46% (1974-1989) and 71% (1997-2002). The risk for those >18 months against ≤12 decreased: hazard ratio (HR); 4.61 and 3.94. For age 13-18 months, EFS increased from 42% to 77%. Outcome was worse if: >18 months (HR 4.47); BM metastases (HR 4.00); and MYCN amplified (HR 3.97). For 1997-2002, the EFS for >18 months with BM involvement and MYCN amplification was 18%, but 89% for 0-12 months with neither BM involvement nor MYCN amplification. CONCLUSIONS There is clear evidence for improving outcomes for children with NB over calendar time. The adverse influence of increasing age-at-diagnosis has declined but it remains a powerful indicator of unfavourable prognosis. These results support the age-of-diagnosis cut-off of greater than 18 months as a risk criterion in the INRG classification system.

Experience with International Neuroblastoma Staging System and Pathology Classification

The International Neuroblastoma Staging System and Pathology Classification were proposed in 1988 and in 1999, respectively, but their clinical value has not yet been fully studied in new patients. Six hundred and forty-four patients with neuroblastoma treated between January 1995 and December 1999 were analysed by these classifications. The 4-year overall survival rate of patients <12 months of age with INSS stages 1, 2A, 2B, 3 and 4S disease was 98.5%, which was significantly higher than the 73.1% rate in stage 4 patients <12 months (P<0.0001). When patients were ⩾12 months, the 4-year overall survival rate of patients with neuroblastoma at 1, 2A, 2B and 3 stages was 100% and that of patients at stage 4 was 48.5% (P<0.0001). As to the International Neuroblastoma Pathology Classification histology, the 4-year overall survival rate was 98.8% in patients with favourable histology and 60.7% in those with unfavourable histology in the <12 months group (P<0.0001). In the ⩾12 months group, the 4-year oral survival of patients with favourable histology was 95.3% and that of patients with unfavourable histology was 50.6% (P<0.0001). Among biological factors, MYCN amplification, DNA diploidy and 1p deletions were significantly associated with poor prognosis in patients <12 months, as were MYCN amplification and DNA diploidy in patients ⩾12 months of age. Multivariate analysis showed that the INSS stage (stage 4 vs other stages) and International Neuroblastoma Pathology Classification histology (unfavourable vs favourable) were significantly and independently associated with the survival of patients undergoing treatment, stratified by age, stage and MYCN amplification (P=0.0002 and P=0.0051, respectively).

RASSF1A hypermethylation in pretreatment serum DNA of neuroblastoma patients: a prognostic marker.

The tumour suppressor gene RASSF1A is known to be frequently silenced by promoter hypermethylation in neuroblastoma tumours. Here we explored the possible prognostic significance of aberrant promoter hypermethylation of RASSF1A in serum DNA samples of patients with neuroblastoma as a surrogate marker for circulating tumour cells. We analysed the methylation status of the RASSF1A gene in matched tumour and pretreatment serum DNA obtained from 68 neuroblastoma patients. Hypermethylation of RASSF1A in tumour samples was found in 64 patients (94%). In contrast, serum methylation of RASSF1A was observed in 17 patients (25%). Serum methylation of RASSF1A was found to be statistically associated with age ⩾12 months at diagnosis (P=0.002), stage 4 (P<0.001) and MYCN amplification (P<0.001). The influence of serum RASSF1A methylation on prognosis was found to be comparable with that of the currently most reliable marker, MYCN amplification on univariate analysis (hazard ratio, 9.2; 95% confidence interval (CI), 2.8–30.1; P<0.001). In multivariate analysis of survival, methylation of RASSF1A in serum had a hazard ratio of 2.4 (95% CI, 0.6–9.2), although this association did not reach statistical significance (P=0.194). These findings show that the methylation status of RASSF1A in the serum of patients with neuroblastoma has the potential to become a prognostic predictor of outcome.