P.A. Voûte

Allelic Loss of Chromosome 1p as a Predictor of Unfavorable Outcome in Patients with Neuroblastoma

BACKGROUND Neuroblastoma is a childhood tumor derived from cells of the neural crest, with a widely variable outcome. Differences in the behavior and prognosis of the tumor suggest that neuroblastoma can be divided into several biologic subgroups. We evaluated the most frequent genetic abnormalities in neuroblastoma to determine their prognostic value. METHODS We used Southern blot analysis to study the allelic loss of chromosomes 1p, 4p, 11q, and 14q, the duplication of chromosome 17q, and the amplification of the N-myc oncogene in 89 neuroblastomas. We also determined the nuclear DNA content of the tumor cells. RESULTS Allelic loss of chromosome 1p, N-myc amplification, and extra copies of chromosome 17q were significantly associated with unfavorable outcome. In a multivariate analysis, loss of chromosome 1p was the most powerful prognostic factor. It provided strong prognostic information when it was included in multivariate models containing the prognostic factors of age and stage or serum ferritin level and stage. Among the patients with stage I, II, or IVS disease, the mean (+/- SD) three-year event-free survival was 100 percent in those without allelic loss of chromosome 1p and 34 +/- 15 percent in those with such loss; the rates of three-year event-free survival among the patients with stage III and stage IV disease were 53 +/- 10 percent and 0 percent, respectively. CONCLUSIONS The loss of chromosome 1p is a strong prognostic factor in patients with neuroblastoma, independently of age and stage. It reliably identifies patients at high risk in stages I, II, and IVS, which are otherwise clinically favorable. More intensive therapy may be considered in these patients. Patients in stages III and IV with allelic loss of chromosome 1p have a very poor outlook, whereas those without such loss are at moderate risk.

N‐myc enhances the expression of a large set of genes functioning in ribosome biogenesis and protein synthesis

The myc oncogenes are frequently activated in human tumors, but there is no comprehensive insight into the target genes and downstream cellular pathways of these transcription factors. We applied serial analysis of gene expression (SAGE) to identify targets of N‐myc in neuroblastomas. Analysis of 42 000 mRNA transcript tags in SAGE libraries of N‐myc‐ transfected and control neuroblastoma cells revealed 114 up‐regulated genes. The majority of these genes have a role in ribosome assembly and activity. Northern blot analysis confirmed up‐regulation of all tested transcripts. Induction was complete within 4 h after N‐myc expression. The large majority of the ribosomal proteins were induced, as well as genes controlling rRNA maturation. Cellular rRNA content was 45% induced. SAGE libraries and northern blot analysis confirmed up‐regulation of many of these genes in N‐myc‐amplified neuroblastomas. As N‐myc can functionally replace c‐myc, we analyzed whether N‐myc targets were induced by c‐myc as well. Approximately 40% of these N‐myc targets were up‐regulated in a c‐myc‐transfected melanoma cell line. These data suggest that myc genes function as major regulators of the protein synthesis machinery.

Clustering of hypermethylated genes in neuroblastoma

CpG‐island hypermethylation of gene promoters is a frequent mechanism for gene inactivation in tumors. Many neuroblastomas have hypermethylation and down‐regulation of CASP8, leading to resistance to tumor necrosis factor–related apoptosis‐inducing ligand (TRAIL). We recently found hypermethylation of the four TRAIL receptors in 9 neuroblastoma cell lines. Here, we analyzed methylation of 34 genes in 22 neuroblastoma cell lines. Of the 29 newly analyzed genes, only FLIP at chromosome band 2q33 was methylated in 8/22 cell lines. The FLIP protein is a negative regulator of Caspase 8. FLIP maps adjacent to CASP8, and their methylation patterns showed a moderate correlation. Furthermore, co‐methylation patterns were observed for the TRAIL receptor pairs DCR1 and DCR2 and between DR4 and DR5. All four receptors co‐localize in chromosome band 8p21. The 6 genes methylated in neuroblastomas appeared to occur in pairs. The genes within each pair have a strong sequence homology and originated from gene duplication. We found no evidence for regional spreading of methylation, given that we did not observe de novo methylation in additional local CpG islands. However, the gene pairs showed a striking co‐regulation at the mRNA expression level. Down‐regulation of FLIP strongly corresponds with down‐regulation of CASP8, and this was also found for DCR1 and DCR2. Only a subset of the down‐regulated genes was methylated. This suggests a mechanism of co‐regulated transcriptional silencing of the gene pairs, followed by a methylation event that is less penetrating. The methylation pattern therefore supports a model in which CpG islands are not randomly targeted by methylation in cancer. Specific transcriptional silencing probably marks genes that can become methylated.

Genetic alterations in childhood medulloblastoma analyzed by comparative genomic hybridization

Despite intensive therapy, the survival of children with medulloblastoma remains disappointing. Moreover, children who survive are affected by serious long-term sequelae of treatment that impair their quality of life. In search of chromosomal aberrations indicative of sites involved in oncogenic transformation and in an attempt to find reliable prognostic markers, the authors analyzed 15 medulloblastomas by comparative genomic hybridization. All neoplasms showed chromosomal abnormalities. The most frequent losses were 17p (7/15 tumors), 8p and 11p (6/15), 10p, 11q, 16q, and 20q (5/15), and 20p (4/15). Gains were recurrently found at 7q (10/15 tumors), 17q and 18q (9/15 tumors), 7p and 13q (7/15), 18p (6/15), and 1q, 4q, 6q, and 9p (5/15 tumors). Four tumors showed loss of 17p together with gain of 17q, suggesting an isochromosome 17q. High-level amplifications were seen at 1p34, 5p15, 13q34, and 18p11 (one tumor each), and at 2p15 in two tumors, one of which was proven to be N-Myc amplification. The overall pattern of alterations found in this study confirms the findings of other studies and adds two novel regions with chromosomal gains, at 13q and 18q. Previous reports on the relation between 17q gain and survival could not be confirmed, whereas amplification of N-myc or L-myc seems to indicate poor clinical outcome.

First line targeted radiotherapy, a new concept in the treatment of advanced stage neuroblastoma

33 previously untreated advanced stage neuroblastoma patients were treated with [131I]meta-iodobenzylguanidine (MIBG). The number of treatments varied between 2 and 7 per patient (mean 3). Toxicity was seldom severe. Only thrombocytopenia WHO-grade 4 was noticed. Response was documented before surgery for the primary tumour was performed. There was one complete response (CR), 18 partial responses (PR), 11 had stable disease (SD) and 3 had progressive disease (PD). After MIBG therapy and surgery, 12 of 33 patients achieved a CR. This approach is feasible, comparable to multidrug chemotherapy in efficacy and less toxic. Long term results are not known yet.

Allelic loss of the short arm of chromosome 4 in neuroblastoma suggests a novel tumour suppressor gene locus

Neuroblastoma is a childhood neural crest tumour, genetically characterized by frequent deletions of the short arm of chromosome 1 and amplification of N-myc. Here we report the first evidence for a neuroblastoma tumour suppressor locus on 4pter. Cytogenetically we demonstrated rearrangements of 4p in 7 out of 26 evaluable tumours (27%). Subsequent analysis of loss of heterozygosity (LOH) by Southern blotting revealed allelic loss of 4p in 16/82 (19.5%) informative neuroblastomas. Taken together cytogenetic and Southern blot analyses showed loss of 4p in 20/86 neuroblastomas analysed (23%). The common deleted region was bordered by the probe D4S 123 and encompassed the distal 34 cM of 4p. We found no evidence for genomic imprinting of the 4p locus as the 4p alleles lost in the tumours were of random maternal and paternal origin. LOH4p was found at all disease stages and in every age group. Furthermore LOH4p was present both in cases with and without LOHIp and amplification of N-myc.

131I-MIBG as a first-line treatment in high-risk neuroblastoma patients

The observed response to 131I-metaiodobenzylguanidine (MIBG) therapy in advanced neuroblastoma after conventional therapy, the non-invasiveness of the procedure, and the high metabolic activity which is frequently observed in untreated tumours led to the concept of substituting 131I-MIBG therapy for combination chemotherapy at diagnosis prior to surgery in patients with advanced disease/high-risk neuroblastoma. The objective of introducing 131I-MIBG therapy as the first therapy in the treatment schedule is to reduce the rumour volume, enabling adequate (>95%) surgical resection of the tumour and to avoid toxicity and the induction of early drug resistance. The advantages of this approach are that the childs general condition is unaffected or improved before it undergoes surgical resection and that chemotherapy is reserved to treat minimal residual disease postoperatively. Thirty-one children who presented with inoperable neuroblastoma (10 Evans stage III, 21 stage IV) were treated according to this protocol. The objective response to the 131I-MIBG therapy at diagnosis with respect to the volume of the primary tumour, the metastases and catecholamine excretion in urine varied from 72 to 81%, which is better than after conventional treatment. Nineteen of 27 evaluable patients (70%) had complete or >95% resection of the primary tumour or did not require surgery at all. Only 11 of 31 patients developed isolated thrombocytopenia and, despite the fact that the bone marrow was invaded in 16 patients, moderate bone marrow depression occurred in only two cases. It is concluded that 131I-MIBG therapy of neuroblastoma at diagnosis is feasible; its effectiveness in attaining operability of the primary tumour is at least equal to that of combination chemotherapy, but its toxicity is considerably less.

A novel local treatment strategy for advanced stage head and neck rhabdomyosarcomas in children: results of the AMORE protocol

The AMORE protocol is a local treatment regimen for head and neck rhabdomyosarcomas (HNRMS), consisting of Ablative surgery, Moulage technique brachytherapy and surgical Reconstruction. The aim of AMORE is to intensify local treatment for children with HNRMS and to avoid external beam radiation therapy (EBRT) and its long-term sequelae. All children with primary irresectable, non-orbital HNRMS in whom EBRT was indicated, were evaluated for the feasibility of AMORE. In 20 children, AMORE was performed (15 with parameningeal disease and five with non-parameningeal disease). Complete remission was achieved in all 20 patients. Local complications were limited. 5 patients experienced a local relapse and 1 patient developed distant metastases. Estimated 5-year OS and EFS were 67.5 and 64.1% for the entire group, and 64.2 and 60.0% for the parameningeal subgroup. We conclude that the AMORE protocol is a feasible strategy, with a good local control rate. Long-term sequelae of EBRT might be avoided although, to date, the follow-up is too short for definitive conclusions regarding these sequelae.

A phase II study of cisplatin, ifosfamide and doxorubicin in operable primary, axial skeletal and metastatic osteosarcoma

Summary Background; Despite advances in the treatment of primary limb osteosarcoma, the outcome of patients with primary metastatic and axial skeletal disease remains poor. The European Osteosarcoma Intergroup have assessed a combination chemotherapy regimen consisting of ifosfamide (IFOS) 3 g/m 2 /dl-2, doxorubicin (DOX) 25 mg/m 2 /dl-3 i.v. bolus and cisplatin (CDDP) 100 mg/m 2 /dl. Patients and methods: One hundred nine previously untreated patients with primary osteosarcoma were registered. Eligibility was confirmed in 103. At presentation, 45 eligible patients had metastatic disease, 15 axial skeletal primary tumours and 43 non-metastatic limb tumours. Results: The major toxicities were myelosuppression (90%, grade 3 or 4) and nausea and vomiting (74%, grade 3 or 4). Overall mean relative dose intensity (RDI) was 80% (88% CDDP, 75% IFOS, 81% DOX). Clinical response as measured by reduction in tumour volume occurred in 36% (95% confidence interval (95% CI): 27%-47%) of primary tumours. Response of pulmonary metastases to chemotherapy was seen in 33% (95% CI: 19%-49%). Good histological response (5=90% necrosis of the tumour) occurred in 33% (95% CI: 22%-45%) of resected tumours. Five-year survival was 62% in limb-non-metastatic, 41% in axial skeletal and 16% in limb metastatic patients. Conclusions: This regimen is active in osteosarcoma but does not appear to be more active than the two-drug CDDPDOX regimen currently recommended by EOI.

Ewing's Sarcoma of the Pelvis: Changes Over 25 Years in Treatment and Results

The pelvic localisations of Ewings sarcoma have the worst prognosis due to large size at diagnosis, frequent distant metastases, radiosensitive organs next to the tumour and difficult surgery. The purpose of the present study was to analyse treatment results over a period of 25 years and to investigate the impact of newer chemotherapy schedules, improved radiotherapy techniques and newer surgical methods on the prognosis. 35 children and young adults were identified from 1967 to 1994 for whom diagnosis, presentation, performed treatment and outcome were available. Tumour size, as measured from CT scans, response to chemotherapy and radiotherapy target volume, could be reviewed in the later years. Actuarial 5-year survival for the whole group was 31% and for the 24 non-metastatic patients 40%, with a disease-free interval of 19%. Tumour size could be measured in 27 patients and ranged from 36 to 1540 cm3. There were 12 local recurrences, 1 in the 4 patients treated with surgery. After 1983, 9 out of 17 irradiated patients developed local failure. 3 patients had adequate fields and one a close field which did not cover completely the prechemotherapy extent and 3 of these recurred. All 4 patients with stable disease after neoadjuvant CT failed locally, not withstanding high-dose radiotherapy. The mean length of neoadjuvant CT tended to be shorter in patients without local relapse. There was no significant difference in survival before and after 1983.