Adela Cañete

Intracranial ependymomas in children: A critical review of prognostic factors and a plea for cooperation

BACKGROUND Current controversies in pediatric intracranial ependymoma include histologic categorization and management. Most of our knowledge of this disease comes from single-institution reports. METHODS A literature search was done, covering the period 1976-1996. The aim of this review is to analyze the prognostic factors reported in the literature over the last 20 years. RESULTS Forty-five series were reviewed, including more than 1,400 children. The largest series reported on 92 patients, and the accrual rate ranged from 0.32-12 patients per year. None of the prognostic factors reported achieved a consensus throughout the different series. Histology remains a major issue, and the range in the incidence of anaplastic ependymo mas (7-89%) highlights the difficulty in agreeing on a histological grading system. The role of surgery on the outcome seems to be determinant. Recent series based on homogeneous imaging-documented extents of resection strongly support the benefit of postoperative radiotherapy. The lack of a proven, effective chemotherapy regimen precludes its use except in prospective pilot studies. CONCLUSIONS Limited information is available from single-institution reports in ependymoma. Only large national or international studies can provide enough information to allow a multivariate analysis of the prognostic factors, and thus lead to new therapeutic proposals.

Poor Survival for Infants With MYCN-Amplified Metastatic Neuroblastoma Despite Intensified Treatment: The International Society of Paediatric Oncology European Neuroblastoma Experience

PURPOSE To report the results of a prospective, nonrandomized European study on infants with neuroblastoma and MYCN gene amplification. PATIENTS AND METHODS Infants with neuroblastoma (stage 2, 3, 4, and 4s) and MYCN gene amplification who were diagnosed between 1999 and 2004 were eligible for enrollment onto the study. After diagnosis, staging, and mandatory biologic studies, induction chemotherapy (IC) with conventional drugs was administered, followed by delayed surgery, megatherapy (busulfan-melphalan as a conditioning regimen), and local radiotherapy. RESULTS Of the 46 infants enrolled onto the study, 35 infants were eligible; of these 35 infants, 97% had metastatic spread (24 infants had stage 4, and 10 infants had stage 4s). Two-year overall survival (OS) was 30% (SE, 0.08), with median survival time of 12 months, and 23 deaths due to disease. Two-year, event-free survival (EFS) was 29% (SE, 0.07). The treatment was well tolerated with no deaths as a result of toxicity or severe toxicity. Despite protocol adherence, 30% of the patients who were assessable for response to IC experienced disease progression or did not respond. Stage and high lactate dehydrogenase reached significance in the univariate analysis (P = .028 and .039, respectively for OS; and P = .05 and .031 respectively, for EFS). Ten of 16 patients who received megatherapy are still alive. CONCLUSION Although treatment was well tolerated, survival was poor and our IC failed to achieve a satisfactory response in 30% of our patients. New therapeutic approaches and more intense world-wide collaboration are needed to achieve a cure in this population.

Excellent outcome with reduced treatment for infants with disseminated neuroblastoma without MYCN gene amplification.

PURPOSE On the assumption that most infants with disseminated neuroblastoma without MYCN amplification (MYCNA) have a favorable prognosis, two concomitant prospective trials were started in which chemotherapy was limited to patients presenting life- or organ-threatening symptoms or overt metastases to skeleton, lung, or CNS. Surgery was to be performed only in the absence of surgical risk factors. PATIENTS AND METHODS One hundred seventy infants with disseminated neuroblastoma without MYCNA, diagnosed between June 1999 and June 2004 in nine European countries were eligible for either of the two studies. Trial 99.2 included all stage 4S infants and those with stage 4 with a primary tumor infiltrating across the midline or positive skeletal scintigraphy who were to be observed in absence of symptoms. Trial 99.3 included infants with overt metastases to the skeleton, lung, and CNS to be treated with a minimum of four chemotherapy courses. RESULTS The 125 infants treated on trial 99.2 had a 2-year overall survival (OS) of 97.6% with no difference between asymptomatic and symptomatic patients (97.7% v 97.3%), patients without or with unresectable primary tumors (96.8% v 100%), and patients without or with positive skeletal scintigraphy without radiologic abnormalities (97.2% v 100%). The 45 infants treated on trial 99.3 had a 2-year OS of 95.6%. No patients died of surgery- or chemotherapy-related complications. CONCLUSION Infants with disseminated disease without MYCNA have excellent survival with minimal or no treatment. Asymptomatic infants with an unresectable primary tumor or positive skeletal scintigraphy without radiologic abnormalities may undergo observation alone.

Excellent Outcome With Reduced Treatment in Infants With Nonmetastatic and Unresectable Neuroblastoma Without MYCN Amplification: Results of the Prospective INES 99.1

PURPOSE To evaluate the efficacy of low-dose chemotherapy in infants with nonmetastatic and unresectable neuroblastoma (NB) without MYCN amplification. PATIENTS AND METHODS Infants with localized NB and no MYCN amplification were eligible in the SIOPEN Infant Neuroblastoma European Study 99.1 study. Primary tumor was deemed unresectable according to imaging defined risk factors. Diagnostic procedures and staging were carried out according to International Staging System recommendations. Children without threatening symptoms received low-dose cyclophosphamide (5 mg/kg/d × 5 days) and vincristine (0.05 mg/kg at day 1; CyV), repeated once to three times every 2 weeks until surgical excision could be safely performed. Children with either one threatening symptom or insufficient response to CyV were given carboplatin and etoposide (CaE), sometimes followed by vincristine, cyclophosphamide, and doxorubicin. No postoperative treatment was to be administered. RESULTS Between December 1999 and April 2004, 120 infants were included in the study. Eighty-eight had no threatening symptoms and 79 received CyV. CaE was given to 49 of them because of insufficient response. Thirty-two children had threatening symptoms, 30 of whom received CaE. Anthracyclines were given to 46 children. Surgery was attempted in 102 patients, leading to gross surgical excision in 93. Relapse occurred in 12 patients (nine local and three metastatic). Five-year overall and event-free survivals were 99% ± 1% and 90% ± 3%, respectively, with a median follow-up of 6.1 years (range, 1.6 to 9.1). CONCLUSION Low-dose chemotherapy without anthracyclines is effective in 62% of infants with an unresectable NB and no MYCN amplification, allowing excellent survival rates without jeopardizing their long-term outcome.

Angiogenesis in Neuroblastoma: Relationship to Survival and Other Prognostic Factors in a Cohort of Neuroblastoma Patients

PURPOSE To study angiogenesis in neuroblastoma, using morphometric and computerized image analysis, and correlate the results with survival and other prognostic factors. PATIENTS AND METHODS Sixty-nine patients from the Spanish Cooperative Study for Neuroblastoma were studied. Tumoral angiogenesis was studied using an avidin-biotin immunoperoxidase technique with an anti-CD34 antibody. Vascular parameters (VPs) were analyzed by a computerized system. Statistical analysis was also performed. RESULTS Sixty-six samples had adequate tumoral tissue, and their tumoral vessels were counted. Endothelial cells were more prominent in pure neuroblastomas than in maturing and more mature tumors. VPs showed no statistical difference between the groups of patients as defined by the levels of the other prognostic factors in neuroblastoma: age, stage, histopathology, TRK-A, P-glycoprotein expression, or MYCN copy number. In patients who relapsed, tumors did not show statistically significant difference in VPs when compared with tumors from patients who did not relapse. There was also no difference in VPs in tumors from living patients when compared with tumors from deceased patients. Overall survival was 75%, and event-free survival was 55% at 50 months. CONCLUSION VPs could be adequately determined by a computerized system in neuroblastoma; however, VPs were not predictive of survival for our patients. In our patients, neither disseminated nor local relapses were influenced by the angiogenic characteristics of the tumors.

Segmental chromosomal alterations lead to a higher risk of relapse in infants with MYCN-non-amplified localised unresectable/disseminated neuroblastoma (a SIOPEN collaborative study).

Background:In neuroblastoma (NB), the presence of segmental chromosome alterations (SCAs) is associated with a higher risk of relapse.Methods:In order to analyse the role of SCAs in infants with localised unresectable/disseminated NB without MYCN amplification, we have performed an array CGH analysis of tumours from infants enroled in the prospective European INES trials.Results:Tumour samples from 218 out of 300 enroled patients could be analysed. Segmental chromosome alterations were observed in 11%, 20% and 59% of infants enroled in trials INES99.1 (localised unresectable NB), INES99.2 (stage 4s) and INES99.3 (stage 4) (P<0.0001). Progression-free survival was poorer in patients whose tumours harboured SCA, in the whole population and in trials INES99.1 and INES99.2, in the absence of clinical symptoms (log-rank test, P=0.0001, P=0.04 and P=0.0003, respectively). In multivariate analysis, a SCA genomic profile was the strongest predictor of poorer progression-free survival.Conclusion:In infants with stage 4s MYCN-non-amplified NB, a SCA genomic profile identifies patients who will require upfront treatment even in the absence of other clinical indication for therapy, whereas in infants with localised unresectable NB, a genomic profile characterised by the absence of SCA identifies patients in whom treatment reduction might be possible. These findings will be implemented in a future international trial.

Hypermethylation of apoptotic genes as independent prognostic factor in neuroblastoma disease

Neuroblastoma (NB) is an embryonal tumour of neuroectodermal cells, and its prognosis is based on patient age at diagnosis, tumour stage and MYCN amplification, but it can also be classified according to their degree of methylation. Considering that epigenetic aberrations could influence patient survival, we studied the methylation status of a series of 17 genes functionally involved in different cellular pathways in patients with NB and their impact on survival. We studied 82 primary NB tumours and we used methylation‐specific‐PCR to perform the epigenetic analysis. We evaluated the putative association among the evidence of hypermethylation with the most important NB prognostic factors, as well as to determine the relationship among methylation, clinical classification and survival. CASP8 hypermethylation showed association with relapse susceptibility and, TMS1 and APAF1 hypermethylation are associated with bad prognosis and showed high influence on NB overall survival. Hypermethylation of apoptotic genes has been identified as a good candidate of prognostic factor. We propose the simultaneous analysis of hypermethylation of APAF1, TMS1 and CASP8 apoptotic genes on primary NB tumour as a good prognostic factor of disease progression. Mol. Carcinog.

The doublecortin gene, a new molecular marker to detect minimal residual disease in neuroblastoma.

Neuroblastoma (NB) is a pediatric cancer of highly variable clinical outcome. Much effort is devoted to detection of minimal residual (MRD) disease through RT-PCR or immunology of tissue-specific markers. Tyrosine hyrdroxylase (TH) has demonstrated a high utility to assess disease dissemination, although this marker can be lost due to clonal variability. Here we propose the use of the doublecortin (DCX) gene as a new molecular marker of neuroblastoma cells. DCX specifically appears in migrating neurons of the central and peripheral nervous system and interacts with and regulates the microtobule cytoskeleton. We have studied this gene by real-time quantitative RT-PCR in a total of 47 primary tumors and 202 samples of bone marrow or peripheral blood from 34 high-risk neuroblastoma patients as well as in 41 normal controls. The expression of DCX demonstrated a good specificity and concordance with TH, showing a higher expression rate in all the sample types studied as well as at different time points from diagnosis. We conclude that DCX would be a more efficient marker of minimal disease in neuroblastoma and perhaps other tumors of neuronal lineage.

Busulfan and melphalan versus carboplatin, etoposide, and melphalan as high-dose chemotherapy for high-risk neuroblastoma (HR-NBL1/SIOPEN): an international, randomised, multi-arm, open-label, phase 3 trial

BACKGROUND High-dose chemotherapy with haemopoietic stem-cell rescue improves event-free survival in patients with high-risk neuroblastoma; however, which regimen has the greatest patient benefit has not been established. We aimed to assess event-free survival after high-dose chemotherapy with busulfan and melphalan compared with carboplatin, etoposide, and melphalan. METHODS We did an international, randomised, multi-arm, open-label, phase 3 cooperative group clinical trial of patients with high-risk neuroblastoma at 128 institutions in 18 countries that included an open-label randomised arm in which high-dose chemotherapy regimens were compared. Patients (age 1-20 years) with neuroblastoma were eligible to be randomly assigned if they had completed a multidrug induction regimen (cisplatin, carboplatin, cyclophosphamide, vincristine, and etoposide with or without topotecan, vincristine, and doxorubicin) and achieved an adequate disease response. Patients were randomly assigned (1:1) to busulfan and melphalan or to carboplatin, etoposide, and melphalan by minimisation, balancing age at diagnosis, stage, MYCN amplification, and national cooperative clinical group between groups. The busulfan and melphalan regimen comprised oral busulfan (150 mg/m2 given on 4 days consecutively in four equal doses); after Nov 8, 2007, intravenous busulfan was given (0·8-1·2 mg/kg per dose for 16 doses according to patient weight). After 24 h, an intravenous melphalan dose (140 mg/m2) was given. Doses of busulfan and melphalan were modified according to bodyweight. The carboplatin, etoposide, and melphalan regimen consisted of carboplatin continuous infusion of area under the plasma concentration-time curve 4·1 mg/mL per min per day for 4 days, etoposide continuous infusion of 338 mg/m2 per day for 4 days, and melphalan 70 mg/m2 per day for 3 days, with doses for all three drugs modified according to bodyweight and glomerular filtration rate. Stem-cell rescue was given after the last dose of high-dose chemotherapy, at least 24 h after melphalan in patients who received busulfan and melphalan and at least 72 h after carboplatin etoposide, and melphalan. All patients received subsequent local radiotherapy to the primary tumour site followed by maintenance therapy. The primary endpoint was 3-year event-free survival, analysed by intention to treat. This trial is registered with ClinicalTrials.gov, number NCT01704716, and EudraCT, number 2006-001489-17. FINDINGS Between June 24, 2002, and Oct 8, 2010, 1347 patients were enrolled and 676 were eligible for random allocation, 598 (88%) of whom were randomly assigned: 296 to busulfan and melphalan and 302 to carboplatin, etoposide, and melphalan. Median follow-up was 7·2 years (IQR 5·3-9·2). At 3 years, 146 of 296 patients in the busulfan and melphalan group and 188 of 302 in the carboplatin, etoposide, and melphalan group had an event; 3-year event-free survival was 50% (95% CI 45-56) versus 38% (32-43; p=0·0005). Nine patients in the busulfan and melphalan group and 11 in the carboplatin, etoposide, and melphalan group had died without relapse by 5 years. Severe life-threatening toxicities occurred in 13 (4%) patients who received busulfan and melphalan and 29 (10%) who received carboplatin, etoposide, and melphalan. The most frequent grade 3-4 adverse events were general condition (74 [26%] of 281 in the busulfan and melphalan group vs 103 [38%] of 270 in the carboplatin, etoposide, and melphalan group), infection (55 [19%] of 283 vs 74 [27%] of 271), and stomatitis (138 [49%] of 284 vs 162 [59%] of 273); 60 (22%) of 267 patients in the busulfan and melphalan group had Bearman grades 1-3 veno-occlusive disease versus 21 (9%) of 239 in the carboplatin, etoposide, and melphalan group. INTERPRETATION Busulfan and melphalan improved event-free survival in children with high-risk neuroblastoma with an adequate response to induction treatment and caused fewer severe adverse events than did carboplatin, etoposide, and melphalan. Busulfan and melphalan should thus be considered standard high-dose chemotherapy and ongoing randomised studies will continue to aim to optimise treatment for high-risk neuroblastoma. FUNDING European Commission 5th Framework Grant and the St Anna Kinderkrebsforschung.

Targeting Neuroblastoma Stem Cells with Retinoic Acid and Proteasome Inhibitor

Background Neuroblastma cell lines contain a side-population of cells which express stemness markers. These stem-like cells may represent the potential underlying mechanism for resistance to conventional therapy and recurrence of neuroblastoma in patients. Methodology/Principal Findings To develop novel strategies for targeting the side-population of neurobastomas, we analyzed the effects of 13-cis-retinoic acid (RA) combined with the proteasome inhibitor MG132. The short-term action of the treatment was compared with effects after a 5-day recovery period during which both chemicals were withdrawn. RA induced growth arrest and differentiation of SH-SY5Y and SK-N-BE(2) neuroblastoma cell lines. Inhibition of the proteasome caused apoptosis in both cell lines, thus, revealing the critical role of this pathway in the regulated degradation of proteins involved in neuroblastoma proliferation and survival. The combination of RA with MG132 induced apoptosis in a dose-dependent manner, in addition to promoting G2/M arrest in treated cultures. Interestingly, expression of stem cell markers such as Nestin, Sox2, and Oct4 were reduced after the recovery period of combined treatment as compared with untreated cells or treated cells with either compound alone. Consistent with this, neurosphere formation was significantly impaired by the combined treatment of RA and MG132. Conclusions Given that stem-like cells are associated with resistant to conventional therapy and are thought to be responsible for relapse, our results suggest that dual therapy of RA and proteasome inhibitor might be beneficial for targeting the side-population of cells associated residual disease in high-risk neuroblastoma.