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Background/aim: Genetic analysis in neuroblastoma has identified the profound influence of MYCN amplification and 11q deletion in patients' prognosis. These two features of high-risk neuroblastoma usually occur as mutually exclusive genetic markers, although in rare cases both are present in the same tumor. The purpose of this study was to characterize the genetic profile of these uncommon neuroblastomas harboring both these high-risk features., Methods: We selected 18 neuroblastomas with MNA plus 11q loss detected by FISH. Chromosomal aberrations were analyzed using Multiplex Ligation-dependent Probe Amplification and Single Nucleotide Polymorphism array techniques., Results and Conclusion: This group of tumors has approximately the same high frequency of aberrations as found earlier for 11q deleted tumors. In some cases, DNA instability generates genetic heterogeneity, and must be taken into account in routine genetic diagnosis.

Purpose: More accurate assessment of prognosis is important to further improve the choice of risk-related therapy in neuroblastoma (NB) patients. In this study, we aimed to establish and validate a prognostic miRNA signature for children with NB and tested it in both fresh frozen and archived formalin-fixed paraffin-embedded (FFPE) samples., Experimental Design: Four hundred-thirty human mature miRNAs were profiled in two patient subgroups with maximally divergent clinical courses. Univariate logistic regression analysis was used to select miRNAs correlating with NB patient survival. A 25-miRNA gene signature was built using 51 training samples, tested on 179 test samples, and validated on an independent set of 304 fresh frozen tumor samples and 75 archived FFPE samples., Results: The 25-miRNA signature significantly discriminates the test patients with respect to progression-free and overall survival (P < 0.0001), both in the overall population and in the cohort of high-risk patients. Multivariate analysis indicates that the miRNA signature is an independent predictor of patient survival after controlling for current risk factors. The results were confirmed in an external validation set. In contrast to a previously published mRNA classifier, the 25-miRNA signature was found to be predictive for patient survival in a set of 75 FFPE neuroblastoma samples., Conclusions: In this study, we present the largest NB miRNA expression study so far, including more than 500 NB patients. We established and validated a robust miRNA classifier, able to identify a cohort of high-risk NB patients at greater risk for adverse outcome using both fresh frozen and archived material., (©2011 AACR.)

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.

Aims: Better understanding of neuroblastoma genetics will improve with genome-wide techniques. However, performing these analyses in samples with <60% neuroblast cells is not adequate. We evaluated the utility of fluorescence in situ hybridization (FISH) on tissue microarrays (TMA) in detecting partial genetic instability (PGI), focusing on samples with ≤50% neuroblast cells., Methods and Results: Alterations of 11q and 17q were detected by FISH on 369 neuroblastoma samples in TMA. Status of the MYCN gene and 1p36 region has been established previously by FISH diagnosis. Partial genetic instability (PGI) was defined as the ratio between segmental genetic alterations detected and number of genetic markers diagnosed in each tumour. Of primary tumours, 14.6% harboured 11q deletions, whereas 42.6% showed 17q gain. PGI was established in 260 primary tumours, 67 of which contained ≤50% neuroblasts. Outcomes were statistically worse for patients whose tumours presented high PGI (P < 0.0001). Multivariate analysis revealed moderate and high PGI as prognostic factors., Conclusions: In the cohort examined in this study, univariate and multivariate analysis confirmed the effect of PGI in patient outcome. PGI established by FISH on TMA is a useful method to identify high-risk patients even if tumours have a cell content of ≤50% neuroblast cells., (© 2011 Blackwell Publishing Limited.)

Telomere length alterations are known to cause genomic instability and influence clinical course in several tumor types, but have been little investigated in neuroblastoma (NB), one of the most common childhood tumors. In the present study, telomere-dependent chromosomal instability and telomere length were determined in six NB cell lines and fifty tumor biopsies. The alternative lengthening of telomeres (ALT) pathway was assayed by scoring ALT-associated promyelocytic leukemia (PML) bodies (APBs). We found a reduced probability of overall survival for tumors with increased telomere length compared to cases with reduced or unchanged telomere length. In non-MYCN amplified tumors, a reduced or unchanged telomere length was associated with 100% overall survival. Tumor cells with increased telomere length had an elevated frequency of APBs, consistent with activation of the ALT pathway. The vast majority of tumor biopsies and cell lines exhibited an elevated rate of anaphase bridges, suggesting telomere-dependent chromosomal instability. This was more pronounced in tumors with increased telomere length. In cell lines, there was a close correlation between lack of telomere-protective TTAGGG-repeats, anaphase bridging, and remodeling of oncogene sequences. Thus, telomere-dependent chromosomal instability is highly prevalent in NB, and may contribute to the complexity of genomic alterations as well as therapy resistance in the absence of MYCN amplification and in this tumor type., (Copyright © 2011 Wiley-Liss, Inc.)

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.

Anticancer monoclonal antibodies (mAbs) targeting specific antigens on the tumour surface are increasingly being applied in cancer treatment. Potential advantages include long half-life, low toxicity, high affinity and specificity. In order to develop novel immune therapies for high-risk cancers, finding tumour targets that are not widely shared by normal cells is a goal. GD2-disialoganglioside is one of them. It is expressed on the surface of a variety of tumours with no curative therapies for patients with advanced disease. In childhood, neuroblastoma is the most common GD2-expressing tumour. Because of this tumour-selective expression, it is an attractive target for tumour-specific therapies such as antibody therapy. Over the last two decades, several anti-GD2 antibodies have been developed. To reduce both toxicity and development of human anti-mouse antibodies (HAMA), research efforts have primarily focused on exploring anti-GD2 antibodies that substitute mouse components by human ones. This review will examine antibodies currently undergoing clinical testing as well as the most recent advances to improve antibody therapy for patients with high-risk neuroblastoma.

There are few reports regarding the presence of retained fixed fragments after removal of indwelling central venous catheters in children. We conducted a retrospective study of 355 patients who underwent removal of central venous catheter from 1996 to 2008. Six patients (1.6%) had a failed attempt of removal, resulting in a remnant of catheter left in the central venous system. All of them had underlying malignant disorders and received chemotherapy for a prolonged period of time. In 2 patients, a second attempt of retrieval was performed that was partially successful. After an average follow-up of 3.5 +/- 1.8 years, 1 patient has developed mild symptoms that could be related to the remaining fragment within the vascular system. Therefore, the incidence of complications caused by retained fixed fragments is low. Patients whose line has been in place for more than 48 months (P = 0.009) and those located in the saphenous vein (P = 0.01) are more prone to experience fragment retention. The decision of retrieval should be balanced according to the presence of symptoms and the length of the fragment retained., (Copyright 2010 Elsevier Inc. All rights reserved.)

Purpose: Solid ovarian tumours are uncommon in childhood. Our aim is to evaluate the outcomes in a single institution over 35 years., Methods: We reviewed their clinical presentation, management, pathology and outcomes from 1972 to 2007., Results: Fifty-three patients, with a median age of 9.2 years (range 0.9-14.2), were registered. Common symptoms at presentation were abdominal pain (75.8%) and abdominal mass (57.4%). Histopathological diagnoses were: 26 mature teratoma, 10 immature teratoma, 8 dysgerminoma, 5 granulosa cell tumours, 2 yolk sac tumours, 1 gonadoblastoma and 1 embryonal carcinoma. Staging (FIGO) for malignant/borderline tumours was: 17 stage I, 1 stage II, 8 stage III and 1 stage IV. Unilateral salpingo-oophorectomy was performed in 47 cases. Sixteen patients underwent chemotherapy after surgery (15 with platinum-based regimen) and postoperative radiotherapy was given in 5 cases. Recurrence was observed in 2 patients and one died (stage III immature teratoma) after a second relapse despite multiple chemotherapy, surgery and radiotherapy. Five-year OS and 5-year EFS were 97% and 94% respectively., Conclusions: Although rare, ovarian tumours must be included in the differential diagnosis of abdominal pain in childhood. Our results confirm their excellent prognosis using conservative surgery and platinum-based chemotherapy. The key point is to maintain excellent outcome while reducing associated morbidity and preserving fertility.

Background: Age at diagnosis is an important risk factor in neuroblastoma (NB) with worse prognosis in children older than 18 months. A more indolent course with long-term relapses and fatal outcome has been described in small series of adolescents. Our objective was to describe biological factors that contribute to this particular behaviour and could be helpful in their treatment., Procedure: NB cases older than 10 years of age at diagnosis registered in the files of the Neuroblastoma Group of SEOP from 1992 to 2007 were included. Disease extension was classified according to the International Neuroblastoma Staging System (INSS). Tumour samples were studied according to the International Neuroblastoma Pathology Classification (INPC). Biological studies included MNA, 1p, 11q and 17q status and ploidy., Results: Twenty-two patients, from 10.1 to 24.6 years old, were included. Advanced stages predominated. 14/17 patients presented unfavourable histology. None had NMA or 1p del. However, 11q del was found in 8/13 cases and 17q gain in 7/11. Overall survival (OS) and event-free survival (EFS) for the entire series at 5 years were 0.45 and 0.32, respectively. Moreover, 5-year OS and EFS for stage 4 patients were 0.33 and 0.15., Conclusions: NB in adolescents is a special subgroup characterised by high-risk prognostic features which differ from those seen in younger patients, especially in relation to genetic abnormalities. The outcome in stage 4 was worse than in younger metastatic children, outlining the need for new therapeutic approaches in this subgroup of patients. The exact cut-off to separate older patients has not yet been established and will probably be based on biology.

Advances in antineoplastic therapy have considerably increased survival rates, and currently in the setting of paediatric population are higher than 70%. Among the potential long-term late effects due to oncological treatment, reproductive dysfunction may have a particularly deep impact on the survivors' quality of life. Until recently, limited fertility preservation options were available before gonadotoxic therapies. In the last few years the development of new assisted reproduction techniques, including ovarian cryopreservation, may be promising in selected groups of patients. Although, the feasibility of these advances in the paediatric and adolescent field have a long way to go, several practical and ethical issues deserve a special review before recommending them for routine medical practice.

Background: More accurate prognostic assessment of patients with neuroblastoma is required to better inform the choice of risk-related therapy. The aim of this study is to develop and validate a gene-expression signature to improve outcome prediction., Methods: 59 genes were selected using an innovative data-mining strategy, and were profiled in the largest neuroblastoma patient series (n=579) to date using real-time quantitative PCR starting from only 20 ng of RNA. A multigene-expression signature was built using 30 training samples, tested on 313 test samples, and subsequently validated in a blind study on an independent set of 236 tumours., Findings: The signature has a performance, sensitivity, and specificity of 85.4% (95% CI 77.7-93.2), 84.4% (66.5-94.1), and 86.5% (81.1-90.6), respectively, to predict patient outcome. Multivariate analysis indicates that the signature is a significant independent predictor of overall survival and progression-free survival after controlling for currently used risk factors: patients with high molecular risk have a higher risk of death from disease and higher risk of relapse or progression than patients with low molecular risk (odds ratio 19.32 [95% CI 6.50-57.43] and 3.96 [1.97-7.97] for overall survival and progression-free survival, respectively, both p<0.0001). Patients at an increased risk of an adverse outcome can also be identified in the current treatment groups, showing the potential of this signature for improved clinical management. These results were confirmed in the validation study, in which the signature was also independently statistically significant in a model adjusted for MYCN status, age, International Neuroblastoma Staging System stage, ploidy, International Neuroblastoma Pathology Classification grade of differentiation, and mitosis karyorrhexis index (odds ratios between 4.81 and 10.53 depending on the model for overall survival and 3.68 [95% CI 2.01-6.71] for progression-free survival)., Interpretation: The 59-gene expression signature is an accurate predictor of outcome in patients with neuroblastoma. The signature is an independent risk predictor, identifying patients with an increased risk of poor outcome in the current clinical-risk groups. The method and signature is suitable for routine laboratory testing, and should be evaluated in prospective studies., Funding: The Belgian Foundation Against Cancer, the Children Cancer Fund Ghent, the Belgian Society of Paediatric Haematology and Oncology, the Belgian Kid's Fund and the Fondation Nuovo-Soldati (JV), the Fund for Scientific Research Flanders (KDP, JH), the Fund for Scientific Research Flanders, the Institute for the Promotion of Innovation by Science and Technology in Flanders, Strategisch basisonderzoek, the Fondation Fournier Majoie pour l'Innovation, the Instituto Carlos III, the Italian Neuroblastoma Foundation, the European Community under the FP6, and the Belgian programme of Interuniversity Poles of Attraction.

Astrocytic neoplasms are genetically heterogeneous; however a low frequency of genomic changes has been found in juvenile pilocytic astrocytoma (PA) in molecular studies. Concerning pleomorphic xanthoastrocytomas (PXA), recent studies have given heterogeneous results for chromosomal alterations. We studied the subtelomeric regions of 19 primary astrocytoma tumors. Results were near normality for the PA group with relative scarcity of chromosomal imbalances, except for the duplication of 3pter in 4/15 and deletion of 21qter in 5/15 of them. In contrast, a specific profile was observed in the 4 PXA tumoral samples. This involved 3pter, 14qter and 19pter duplication and 4qter, 6qter, 9qter, 13cen, 17pter, 18qter and 21qter deletion. Our results indicate that the chromosomal and genetic aberrations in PXAs differed from those typically associated with the diffusely infiltrating astrocytic and oligodendroglial gliomas. These genetic differences would likely contribute to the more favorable behavior of PXAs and may be helpful for molecular differential diagnosis of pediatric cerebral tumors.

Purpose: Neuroblastoma is an embryonal tumor of neuroectodermal cells. Patients with metastatic neuroblastoma have a poor survival rate, which has led to numerous efforts to develop prognostic markers. Cancer/testis-specific antigens MAGE-A1 and MAGE-A3 genes were proposed as minimal residual disease (MRD) markers in neuroblastoma, but its usefulness for this purpose is rather limited., Methods: We studied 47 primary neuroblastoma tumors. RNA was extracted and cDNA was prepared by reverse transcription. Detection of the MAGE-A1 expression was done by hybridization of the RT-PCR products. We used methylation-specific-PCR to perform the epigenetic studies., Results: We studied the MAGE-A1 and MAGE-A3 expressions, and the MAGE-A1 expression showed significant association with tumor stage, absence of bone marrow infiltration and survival. A multivariate analysis enabled us to conclude that the MAGE-A1 expression represents a new independent predictive factor, which is independent of N-Myc amplification (P value = 0.000), age at diagnosis (P value = 0.002) or tumoral stage (P value = 0.024). Considering the epigenetic regulation of MAGE-A1, we analyzed its methylation profile, and found a significant association with its expression in tumor cells. Moreover, we found tumors that failed to show the MAGE-A1 expression despite the hypomethylated sequence, and corresponded to advanced neuroblastoma that might share another mechanism involved in MAGE-A1 silencing. Given the association described between genome-wide hypomethylation and microsatellite instability, we determined the MSI status of tumor samples, finding a significant correlation with the MAGE-A1 expression and, more specifically, with the hypomethylated status of this gene only in female patients., Conclusion: We conclude that the MAGE-A1 expression is associated with good prognosis in neuroblastoma.

Background: Neuroblastic tumors (NT) are pediatric neoplasms with a heterogeneous genetic profile. They present genotypic alterations of prognostic value, the study of which is mandatory in designing therapeutic management. Tissue microarrays (TMA) from paraffin material allow the analysis of a large number of cases with minimal costs. The main purpose of the present study is to analyze specific genetic markers of neuroblastic tumors included in TMAs and determine their prognostic value. We compare the results obtained by different molecular techniques at different substrates to evaluate the feasibility of these assays., Procedure: One hundred thirty-nine samples were included in four different TMAs. We performed FISH assays to determine the status of MYCN gene, 1p36 region and 17q23 arm. The prognostic value of the genetic markers as well as the statistical correlation among clinical variables and outcome were analyzed by SPSS., Results: MYCN amplification was detected in 35.3% of the cases, whereas 1p36 deletion and 17q23 gain was observed in 46.8% and 58.3% of the cases, respectively. An adverse prognosis was noted among these patients. Other adverse factors were age (>18 months) as well as high stage of disease (stage 4). Phenotypic signs of differentiation correlated with good outcome., Conclusion: Retrospective studies using paraffin-embedded tissues assembled in TMA are a useful tool for the analysis of prognostic factors in NT., ((c) 2008 Wiley-Liss, Inc.)

Introduction: Posttransplant lymphoproliferative disorder (PTLD) constitutes a heterogeneous group of diseases. We summarize the experience of our hospital, one of Spain's largest series of renal (294), liver (47) and allogeneic stem cell transplants (67), where four cases of PTLD have developed related to complex viral infections., Methods: Case 1 was a 24-month-old boy diagnosed with acute lymphoblastic leukemia who underwent allogeneic stem-cell transplantation (SCT). He was seropositive for Epstein-Barr virus (EBV) and developed an aggressive Bcell non-Hodgkin's lymphoma (B-NHL) related to EBV reactivation and human herpesvirus 6 (HHV-6) infection. Cases 2, 3, and 4 developed after kidney transplantation and were all EBV seronegative. Case 2 had associated cytomegalovirus (CMV) and EBV infection. Cases 3 and 4 only revealed EBV viral load. Cases 1, 3, and 4 progressed rapidly, with fatal outcome. Global incidence of PTLD in our series is 1.1%., Conclusion: PTLD is a rare but life-threatening condition. Although EBV plays a clear role in its pathogenesis, other associated viral infections could trigger this situation. Current therapies include rituximab, decreasing immunosuppressive drugs. and conventional chemotherapy.

Between 1978 and 2006, the European Group for Blood and Marrow Transplantation registered 4098 high-dose therapy (HDT) procedures followed by stem cell rescue (SCR) (3974 autologous/124 allogeneic) in patients with neuroblastoma. The 5-year rates for overall (OS) and event-free survival are 37 and 32%, respectively. The median age at diagnosis is 3.9 years (0.3-62 years) with 76 patients older than 18 years. Patients above 10 years carry a 2.5-fold higher risk. Younger patients cure significantly (<0.001) better with OS rates of 40 and 30% for age groups 2-4 years and 4-10 years, respectively. Their risks are about twofold higher than that of patients below 2 years with OS rates of 60%. The better the quality of remission status before HDT/SCT the better are the observed OS rates: 43% in CR1 (1199 patients) and 42% for CR2 (140 patients), and 36% for those in very good partial or partial remission (1413 patients) and 21% for those with sensitive relapse (134 patients). Patients reported with stable disease in first remission still had an OS rate of 30%. Multivariate analysis shows significantly better OS in the age group of less than 2 years (<0.0001), as well as a better quality of remission status before HDT/SCT (P<0.0001), with the use of peripheral stem cells (P=0.014), autologous SCT (P=0.031) and busulphan/melphalan HDT (P<0.001). Busulphan/melphalan HDT/SCT in first remission achieves an OS of 48%, while it is only 35% with other regimens (P<0.001), including melphalan alone, other melphalan-containing regimens, a variety of other drugs given as a single HDT as well as the addition of TBI or sequential HDT/SCT procedures. Further progress in the field may only be expected from large-scale international randomized trials.

Neuroblastoma is one of the most frequently occurring solid tumours in children, especially in the first year of life, when it accounts for 50% of all tumours. It is the second most common cause of death in children, only preceded by accidents. The most peculiar characteristic of neuroblastoma is its clinical heterogeneity. Approximately half of the cases are classified as high risk, with overall survival rates around 40% despite intensive multimodal therapy. Nevertheless, other subsets of neuroblastomas will undergo spontaneous regression and others will show very slow progression. Despite many advances in the past three decades, neuroblastoma has remained an enigmatic challenge to clinical and basic scientists. Elucidation of the exact molecular pathways of neuroblastoma will enable researchers and clinicians to stratify the disease and adapt therapy to the risk of relapse or progression. This review focuses on recent advances in our understanding of the biology of this complex paediatric tumour. Neuroblastoma is already one of the first examples for the use of tumoral genetic markers as a tool for defining tumour behaviour and to aid clinical staging.

Introduction: The information offered by the new genomic and proteomic techniques will play a central role in our knowledge of cancer; but it is limited by the lack of available tissue samples. Cancer in children is a sum of infrequent diseases, so tumor banks are support tools for translational research, providing access to a sufficiently large series of samples, which would minimize the asymmetric effect of the diverse origin., Material and Methods: From 2003 a Molecular Pathology Network in Pediatric Solid Tumors Netwoks exists in Spain, and we are a part of it. Our aim was to create a pediatric tumor bank program and consensus documents about its use., Results: Standard Operating Procedures for collection and transport of samples have been created., Conclusions: Thinking about the fast progress in Molecular Biology and the low frequency in pediatric tumors, it is vital to consider the importance of a bio bank.

In addition to clinical and biological factors, further valuable prognostic information in neuroblastoma (Schwannian stroma-poor) (NB) patients is provided by the histopathologic analysis and the application of the International Neuroblastoma Pathology Classification (INPC) system. The objective of this study was to assess the prognostic impact of the INPC classification in a series of NB (Schwannian stroma-poor) and its relation with other prognostic factors. One hundred eighty-two cases of NB were collected from the files of the Spanish Neuroblastoma Registry. Slides were reviewed, and NB cases were grouped into favorable and unfavorable categories according to INPC criteria, taking into account morphological features (mitosis-karyorrhexis index, histological subtype) and patient's age at diagnosis. Other pathological [presence of calcifications, tissular components, and number of mitotic cells per 10 high-power field (HPF)], immunohistochemical (P-glycoprotein and Ki-67 protein expression) and genetic (MYCN amplification and chromosome 1p deletion) features were also studied. Statistical analyses of overall survival with Kaplan-Meier curves and a multivariate study using Cox regression were performed (40.3% of NBs were considered favorable and 59.7% unfavorable). Unfavorable NB showed a mean survival time of 57 months compared with 89 months in favorable cases. Advanced stage, more than ten mitoses per 10 HPF, Ki-67 expression in more than 30% of tumor cells, MYCN oncogene amplification and chromosome 1p deletion were observed more frequently in unfavorable NB. The Cox regression analysis demonstrated that clinical stage (International Neuroblastoma Staging System stage 4) and histological subtype (undifferentiated NB) were the most important factors that influence the overall survival (p<0.001). INPC classification results are major prognostic indicators in NB and should be considered in the therapeutic stratification of NB patients.

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.

Neuroblastoma and pheochromocytoma have the same embryonal origin. They originate from neural crest cells, and they usually affect suprarenal glands. The SDHB gene encodes the B subunit of succinate dehydrogenase, a protein implicated in the electron transport chain and Krebs cycle. Some mutations have been described in this gene in pheochromocytoma, and this gene could be an appropriate candidate for its study in neuroblastoma given its localization in 1p35-36. The aim of this study was to analyze neuroblastoma tumors in order to assess a possible implication of this gene in neuroblastoma development. We studied 28 neuroblastoma tumor samples from different stages. Mutation research in genomic DNA was carried out after individual amplification of each of the eight SDHB exons by SSCP analysis and sequencing of those samples with migration pattern variants. No variant was found except for three polymorphisms in four neuroblastoma samples. The first polymorphism was a synonymous A-->C change in the third position of codon 6 (exon 1). The other two polymorphisms were a TTC insert at the 5' flanking intron sequence of exon 5 in a stretch of seven TTC repeats. Upon the basis of posterior microsatellite instability and hypermethylation promoter studies, which were not significant, we can conclude that the SDHB gene, a positional candidate gene, is unlikely to be related to either initiation or tumoral progression in neuroblastoma.

Neuroblastoma is the most common solid tumour in childhood. Modern management includes a biopsy to perform genetic studies. Based on clinical data and Myc-N amplification (MNA), patients are divided in three prognostic groups: the low-risk (Stage 1, 2, 4S without MNA) has an event-free survival (EFS) of > 90% with surgery alone; the intermediate-risk (Stage 3, > 1 year of age, without MNA and Stage 3 and 4 infants without MNA) has an EFS of approximately 80% with mild chemotherapy and surgery; the high-risk group includes Stage 4, > 1 year of age and any stage and age with MNA. These patients are treated with chemotherapy, surgery, megatherapy, irradiation and 13-cis-retinoic acid. With this complex therapy, a 5-year EFS of 30-50% can be obtained.

We assessed the utility of GAGE gene expression as a marker of minimal residual disease (MRD) in neuroblastoma. The GAGE gene family shows a high degree of homology (>90%), clustering into two subgroups. GAGE-1, -2, and -8 form one subset, almost identical among themselves, while GAGE-3 to -7 constitute the other subset. The entire GAGE family (GAGE-1-8) was studied by RT-PCR followed by Southern blotting to increase both the sensitivity and specificity of the technique. Surprisingly, expression of GAGE was detected in 59% of peripheral blood samples from normal controls (20/35) as well as in a similar proportion from neuroblastoma patients with localized disease (stages 1 and 2). The study of GAGE-1, -2, and -8 with specific primers lowered this percentage to 28% (10/35), of which only two (6%) showed a high level of expression (directly visualized after RT-PCR). We conclude that GAGE genes can show a variable, usually low level of illegitimate expression in normal blood cells, and therefore their use as MRD markers should be taken with caution.

Langerhans cell histiocytosis (LCH) is a granulomatous disease which can involve multiples sites of the body. Diagnostic imaging is of utmost importance in the management of these patients. Up to now radiographic skeletal survey and bone scintigraphy (BS) have been used to assess bone involvement (both with low specificity). Magnetic resonance imaging (MRI) and CT have been used to assess visceral involvement but with the limitation that they cannot give information about the functional status. Recently somatostatin receptor scintigraphy (SSRS) has been proposed to detect active lesions and to monitor response to treatment. The aim of this study is to assess bone and somatostatin receptor scintigraphy in the detection of bone involvement in LCH in children. Twenty scintigraphies (12 SSRS and 8 BS) were performed in seven patients (3 girls and 4 boys) aged at diagnosis: 18 month-12 years (mean age 6 years). The findings obtained in the scintigraphies were compared with clinical evolution and other imaging techniques. Bone scintigraphy detected all the LCH bone lesions, and discovered one unknown lesion. SSRS scintigraphy visualised the active lesions in 3 patients (clinical and other imaging techniques were also positive). SSRS was negative in one patient classified as disease free and another in clinical remission. SSRS detected 2 new unknown bone lesions, but could not detect LCH bone lesions confirmed in other imaging techniques in 2 patients. Somatostatin receptor and Bone scintigraphy can be used to detect active LCH bone lesions in children and can help to monitor response to treatment. Further studies with more patients are needed to confirm the diagnostic usefulness of these techniques.

To evaluate factors influencing outcome and incidence of long-term complications, we analyzed, in a retrospective, multicenter study, 387 children who underwent autologous hematopoietic stem cell transplantation (HSCT) for acute myeloid leukemia (AML) in first complete remission (CR). Median follow-up time from transplantation was 60 months. Transplantation of bone marrow cells was performed in 318 children, whereas in 60 patients peripheral blood progenitor cells (PBPCs) were used. In multivariate analysis, we investigated the variables influencing probability of hematopoietic recovery, transplantation-related mortality (TRM), relapse, and leukemia-free survival (LFS). We found that use of PBPCs as stem cell sources and use of BCNU (N,N-bis[2-chloroethyl]-N-nitrosourea), amsacrine, VP-16, and cytosine arabinoside (BAVC) as a preparative regimen were associated with faster neutrophil recovery. Infusion of PBPCs, young age of patients, use of BAVCs, and absence of marrow purging predicted an accelerated platelet reconstitution. The 5-year Kaplan-Meier estimates of TRM, relapse, and LFS were 3% +/- 1%, 39% +/- 3% and 60% +/- 3%, respectively. Relapse probability was increased in children given the BAVC regimen, and it was decreased after in vitro purging of hematopoietic progenitors and in children with a French-American-British classification of M3 and a time interval of 170 days or more between CR and HSCT. These 2 latter variables favorably influenced the probability of LFS, which was, by contrast, reduced with the BAVC regimen. Thirty-three percent of patients surviving more than 18 months experienced at least one late sequela; use of total body irradiation was the only predictive factor. The results obtained in this analysis can be of help in designing prospective studies of autologous HSCT in children with AML in first CR.

Stage 4S neuroblastoma is a disease associated with spontaneous regression and good survival. We present a patient whose evolution has shown the variety and complexity of this disease in infants. Biologic factors, such as ploidy, MYCN copy number, loss of 1p36, and other chromosomal gains and losses were determined. A complex pattern of genetic abnormalities, such as near-diploidy, MYCN gain (2-4 copies per haploid genome) and imbalance/deletion of 1p36 was seen in the diagnostic sample. An extensive disseminated disease after a latent period of 26 months was associated with a special genetic evolution, such a tetraploidy, MYCN amplification (2:100-500 copies), 1p36 deletion, and gain of 17q. Our results provide evidence that either the primary tumor was heterogeneous in terms of gene amplification or that amplification was acquired later on as a transition from MYCN gain. We suggest that near-di-/tetraploid 4S tumors with MYCN gain and/or deletion 1p could be progressing 4S tumors.

Peripheral neuroblastic tumors (PNT), are heterogeneous neoplasms that include neuroblastoma (NB), ganglioneuroblastoma (GNB) and ganglioneuroma (GN) and present great biological heterogeneity. There are few reports analyzing PCNA and Ki-67 expression in PNT; however, controversy exists concerning the specificity of PCNA as a real proliferative marker. The objective of our study was to determine which of these cellular proliferation markers is more specific on cellular cycle and could contribute with more information on the cell cycle. We found that PCNA was expressed in NB unfavourable cases, with MYCN amplification and high mitosis-karyorrhexis-index (MKI). Whereas, Ki-67 showed statistical significance regarding cases unfavourable with intermediate and high MKI, aneuploid and stages 3 and 4. Survival showed that patients with tumor not expressing Ki-67 (MIB1) lived longer than those without PCNA (88.93 vs 74.05%). We conclude that Ki-67 expression permits reliable detection of the cellular proliferation neuroblastoma fraction and provides useful prognostic information when associated with other biological factors.

Background/purpose: The importance of primary tumor resection in stage IV neuroblastoma is controversial. The authors analyzed prospectively the role of surgery in a multicentric series of stage IV neuroblastoma patients., Methods: Patients were studied according to the International Neuroblastoma Staging System (INSS) recommendations. Age, sex, location of the tumor, type of metastases, time of resection (initial or delayed), extension of resection, surgical complications, pathology, N-myc and Shimada classification results, relapses, and outcome were studied. After diagnosis, children received induction chemotherapy followed by delayed surgery and autologous stem cell transplantation or maintenance chemotherapy. Resection was classified as complete (C), greater than 90% (P1), greater than 50% (P2), less than 50% (P3), and biopsy (B)., Results: Ninety-eight stage IV children were admitted in the study from June 1992 to July 1999. Seventy-six were older than one year, and in 78 the primary tumor was abdominal. Bone was the most common metastatic site followed by bone marrow. Initial biopsy was performed in 74 patients, and resection in 6, with one complication in each group. N-myc was amplified in 20 of 80 tumors, and Shimada was unfavorable in 45 of 67. Delayed surgery was performed in 70 cases, achieving gross total resection in 55 (79%); there were minor complications in 10%. Mean survival rate time was 50 months. Event-free survival rate (EFS) at 5 years for the entire series is 0.32, but 0.0 for children having biopsy only, 0.25 for less than 50% resection, 0.31 for 50% to 90% resection, 0.44 for greater than 90% resection, and 0.33 for complete resection. Differences were statistically significant only when compared with the biopsied group. EFS rate for infants was 0.56, but, again, there was no difference in relation to the type of resection. There were 46 relapses, 12 of them local, 7 of 20 N-myc-amplified tumors, and 4 of 60 not amplified (P <.005)., Conclusions: Biopsies of stage IV neuroblastoma allow safe assessment of N-myc and other biological factors on tumor tissue. Delayed surgery after chemotherapy is performed with a low rate of complications, achieving a good local control of disease. N-myc-amplified tumors have higher local relapse rates than nonamplified and therefore would need more intensive local treatment. The final outcome in these patients is determined more by metastatic relapses than by the degree of resection., (Copyright 2002, Elsevier Science (USA). All rights reserved.)

Background: Our objective was to study the proliferation and apoptotic process in 111 cases of neuroblastoma (NB) and to seek their relationship with other prognostic factors and survival., Methods: Immunohistochemistry following ABC peroxidase was carried out for PCNA, Ki-67, bcl-2, and p53 proteins. Apoptosis analysis was performed with in situ detection of chromosomal breakdown. Molecular detection of DNA ladders by electrophoresis and amplification of MYCN was studied with PCR and Southern blot. Statistical study was performed with Pearson chi(2) and Kruskal-Wallis tests and Cox regression., Results: Our results indicate that proliferative factors PCNA and Ki-67 were correlated to each other as well as to advanced stage and MYCN amplification. Regarding apoptosis, we found expression of bcl-2 protein in cases of NB without differentiation and advanced stages. p53 protein showed an inverse relation with bcl-2 and cell death measured by assay protein. In situ determination of apoptosis was found mainly in differentiated and stage 4s cases. Multivariate analysis revealed protein as the most independent risk factor of our study., Conclusions: The study of cellular proliferation and apoptosis contributes with information of prognostic value that could be applied to the design of different protocols for treatment of neuroblastoma.

Background: Stage 4 and MYCN amplified (MNA) neuroblastoma in children have a poor prognosis. Our aim was to increase initial and long-term response in this population., Procedure: High-risk children were studied according to the International Neuroblastoma Staging System, then treated with high-dose cyclophosphamide and high-dose carboplatin, followed by surgery and autologous stem cell transplant or maintenance chemotherapy., Results: From June 1992 to December 1998, 83 children were admitted in the study (72 stage 4> 1 year, 5 stage 4 MNA infants, and 6 MNA stage 3 children); tumor tissue was obtained from 73, MYCN was performed in 65, being amplified in 21 (32%). Induction chemotherapy was administered in the expected time in 35% of patients. Its toxicity was mainly hematologic followed by infections, and there were 3 chemotherapy-related deaths. Delayed surgery was performed on 60 patients with complete or >90% resection in 80% of cases. Chemotherapy plus surgery produced some response in 90% of patients, 53% were in CR/VGPR; 49 children received autologous SCT, and 16 received maintenance chemotherapy for 9 months. Follow-up ranges are 1-87 months, mean 30 months. S and EFS at 4 years are 0.33 (SD 0.02)., Conclusions: High-dose cyclophosphamide and high-dose carboplatin are effective in the initial treatment of neuroblastoma; combined with surgery they produce some response in most patients. Nevertheless, the CR/VGPR rate reaches only 53%. Survival time has also been prolonged but most patients relapse with metastases., (Copyright 2001 Wiley-Liss, Inc.)

A 10-year-old girl with M2 acute myeloid leukemia underwent an unrelated cord blood transplantation in refractory first relapse. On day +13, after 48 hours with fever, she showed a measles-like rash, and on day +15, she began experiencing neurologic symptoms (headache, tremors, weakness, nystagmus, mild confusion, speaking, taste, and behavior disturbances, and focal seizures). She also had amnesia for recent events with disability to learn, mimicking Wernicke-Korsakoff syndrome. Computed tomography of the brain and cerebrospinal fluid (CSF) and electroencephalogram were nonspecific. We found human herpesvirus 6 (HHV-6) DNA in CSF and cytomegalovirus in bronchoalveolar lavage using polymerase chain reaction techniques. Treatment with ganciclovir and foscarnet was effective, with total resolution of symptoms.

Purpose: Infants with acute leukemia have a poor prognosis when treated with conventional chemotherapy. It is still unknown if stem-cell transplantation (SCT) can improve the outcome of these patients. In the present study, we review our experience with SCT in infant acute leukemia to clarify this issue., Patients and Methods: We report the results of 26 infants who were submitted to a SCT for acute leukemia. There were 15 cases of acute myeloid leukemia and 10 cases of acute lymphoid leukemia. One patient had a bilineal leukemia. Twenty-two patients were in their first complete response (CR1), three were in their second CR, and one was in relapse. Eight patients were submitted to allogeneic SCT, and 18 underwent autologous SCT., Results: With a median follow-up of 67 months, the 5-year overall survival and disease-free survival (DFS) are 64% (SE = 9%) and 63% (SE = 10%), respectively. Autologous and allogeneic SCT offered similar outcome. There was not any transplant-related mortality, and all deaths were caused by relapse in the first 6 months after SCT. In multivariate analysis, the single factor associated with better DFS was an interval between CR1 and SCT of less than 4 months (P: <.025)., Conclusion: SCT is a valid option in the treatment of infant acute leukemia, and it may overcome the high risk of relapse with conventional chemotherapy showing very reduced toxicity. This study suggests that SCT should be performed in CR1 in the early phase of the disease.

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.

Objective: Central nervous system (CNS) tumors are the most frequent solid tumors in children. Twelve to twenty percent are diagnosed in patients younger than two years of age and these patient present more morbidity and mortality due to the illness and the treatment itself., Patients and Methods: A retrospective study of CNS tumors in children younger than three years of age diagnosed in our hospital between 1985 and 1995 was carried out., Results: We treated 21 patients between 1985 and 1995. There were 10 male and 11 females. The mean age was 20.3 months (range: 0-32 months). The mean time between symptoms and treatment was 2.4 months (range: 0-18 months). The most common symptoms included ataxia, nausea and vomits. The most common locations of the tumor were: infratentorial (57.1%) and supratentorial (38.1%). Complete surgery was performed in 3 patients, subtotal in 10, partial in 5, and a biopsy in 2. The anatomical-pathological diagnosis was: astrocytoma (6), ependinoma (5), meduloblastoma (4), ganglioglioma (1), neuroblastoma (1), and primitive neuroectodermic tumor (1). We could not document the histology in 3 patients. Ten patients received chemotherapy that was well tolerated and 14 received radiotherapy whose sequels were updated. The mean follow-up period was 44.42 months (range: 0-136 months). Overall survival was 42.86%. There were no statistically significant differences in survival between those who were irradiated and those who were not, nor between those with supra-or infratentorial tumors., Conclusions: CNS tumors in children younger than three years of age have a worse prognosis than in older children. New therapeutic schedule with chemotherapy are being tested to avoid radiotherapy side-effects.

Background: Prognosis in neuroblastoma is based upon several clinical factors such as age, tumoral staging and other genetic factors like N-myc oncogene amplification or deletion of the short arm of chromosome 1 (del 1p). Recent reports indicate that bcl-2 protein expression is associated with a poor outcome in patients with neuroblastoma., Materials and Methods: We present a study of 80 cases from the files of the Spanish neuroblastoma study group (N-II-92) analysing bcl-2 protein expression by means of immunohistochemical methods and its relation with other parameters such as histopathology, PCNA expression, N-myc amplification and DNA study of apoptosis., Results: We found a statistical correlation between bcl-2 protein expression and unfavourable histopathology, N-myc amplification and PCNA nuclear staining. An inverse relation between bcl-2 staining and apoptosis was detected., Conclusions: Based on present findings it can be concluded that the determination of bcl-2 protein provides prognostic information when associated with other biological factors involved in neuroblastomas.

The records of 116 patients from a single center (1970-1993) with newly diagnosed Ewing's sarcoma or primitive neuroectodermal tumor were reviewed retrospectively. The aim of this study was to ascertain the impact of pretreatment variables on disease-free survival. Median age was 14 years (range 1-34). Twenty patients presented with metastatic disease. Treatment consisted of systemic multiagent chemotherapy plus local irradiation (39%), wide resection (22%), or both (35%). Median potential follow-up was 10.7 years (range 2-26). Three patients developed second malignancies (1 breast carcinoma, 2 acute myeloid leukemias). Median time to relapse was 24 months (range 3-143). The actuarial disease-free survival was 37.4% at 5 years, 33.3% at 10 years and 27.8% at 15 years. Neoadjuvant chemotherapy and a therapy-induced tumor necrosis > or = 90% were associated with a better outcome. Patients undergoing surgical resection had a superior disease-free survival than those treated without surgery (45 vs. 18% at 10 years, p = 0.0009). Multiple regression analysis showed that raised serum lactate dehydrogenase levels (p < 0.001), hypoalbuminemia (p = 0.001) and distant metastases at diagnosis (p = 0.03) were independent adverse prognostic factors. In conclusion, one third of patients with Ewing's sarcoma become long-term survivors with combined modality treatment. Late relapses and second neoplasms are of concern. Prognostic factors should be considered in the selection of therapy, and the value of serum albumin warrants confirmatory studies.

Common origin of sympathoblasts and pheochromoblasts from the neural crest cells is generally accepted. Neuroblastoma and pheochromocytoma result from an abnormal proliferation of these committed cells. They are included in the group of neuroendocrine neoplasms, formerly named Apudomas. Previous reports of mixed tumours of neuroblastoma and pheochromocytoma, and ganglioneuroma and pheochromocytoma, support this hypothesis. A case of extra-adrenal pheochromocytoma in an adolescent who 15 years before was successfully treated for a stage IV-S neuroblastoma without evidence of primitive tumour is reported. Two hypothesis are formulated: (1) the second neoplasm may have arisen from the unlocated primitive tumour, probably silent evolution could be due to its maturation into ganglioneuroma-pheochromocytoma; (2) taken into account a common origin of both neoplasms, the patient may have a genetic predisposition to develop neuroendocrine tumours. Whatever the mechanism, second neoplasms, as the case reported, late relapses, and late toxic effects may justify long follow-up of neuroblastoma survivors.

This survey includes 25 children with Wilms' tumor undergoing high-dose chemotherapy associated with autologous bone marrow transplantation (ABMT) in the period June 1984-December 1991 and enrolled in the European Bone Marrow Transplantation Registry for Solid Tumors. At diagnosis, 12 children presented stage IV disease, 5 stage III, 3 stage II, and 5 stage I. Before ABMT, 21 children had 1 to 4 relapses (median 1); 13 achieved a second or subsequent complete remission (CR), four stage IV children failed to respond to first line treatment and achieved either CR (3 patients), or partial remission (PR) after second line therapy. At high-dose chemotherapy, 17 children were in CR and 8 had measurable disease. Seven different high-dose regimens were administered, even if 20 children received melphalan mostly associated with vincristine and 8 involved field radiotherapy. Three children died early of pneumonitis; 2 developed an acute transient renal failure, 1 a chronic renal failure. Out of the 8 children with target disease at graft, 2 died of toxicity, 5 achieved CR, 1 obtained PR, and only 1 is presently alive in CCR at 39 months after ABMT. Of the 17 children grafted in CR, 8 are alive event-free at 14-90 months (median 34) from ABMT; 7 relapsed at 3-23 months (median 7 months); 1 died of toxicity and 1 was lost to follow-up in CR at 12 months. A salvage attempt with high-dose chemotherapy in children with resistant or poor prognosis recurrent Wilms' tumor seems to be justified. An international cooperative protocol taking into account the increased risk of lung and renal toxicity is necessary.

Some cases of conversion from acute lymphoblastic leukemia (ALL) to acute nonlymphoblastic leukemia (ANLL) at relapse have been reported recently. We report three cases initially diagnosed as having ALL and showing morphological, cytochemical, and immunophenotypic features of ANLL at relapse (lineage switch). Conversion was observed among 14 patients who developed bone marrow relapse while undergoing intensive treatment with our ALL protocol, which includes teniposide, and that had been administered to 62 patients. The three cases converted at first relapse, with a mean time of 20 months (13-29 months). Clinical and immunologic characteristics of T-cell leukemia were present in one patient. Changes documented in cytogenetic studies are discussed. The underlying mechanisms for the lineage switch remain unclear as does its relation with mixed lineage leukemias, but we believe that drugs employed in our therapy protocol could have had an influence on this conversion.