Central nervous system relapse in high-risk stage 4 neuroblastoma: The HR-NBL1/SIOPEN trial experience.

BACKGROUND: There is rising concern on the impact of new strategies, such as high-dose chemotherapy (HDC) and immunotherapy, on the pattern of relapse in high-risk neuroblastoma (HR-NBL). Our aim is to evaluate the incidence and identify risk factors for first recurrence in the central nervous system (CNS) in HR-NBL. PATIENTS AND METHODS: Data from patients with stage 4V HR-NBL included from February 2002 to June 2015 in the prospective HR-NBL trial of the European International Society of Pediatric Oncology Neuroblastoma Group were analysed. Characteristics at diagnosis, treatment and the pattern of first relapse were studied. CNS imaging at relapse was centrally reviewed. RESULTS: The 1977 included patients had a median age of 3 years (1 day-20 years); 1163 were boys. Among the 1161 first relapses, 53 were in the CNS, with an overall incidence of 2.7%, representing 6.2% of all metastatic relapses. One- and three-year post-relapse overall survival was 25 ± 6% and 8 ± 4%, respectively. Higher risk of CNS recurrence was associated with female sex (hazard ratio [HR] = 2.0 [95% confidence interval {CI}: 1.1-3.5]; P = 0.016), MYCN-amplification (HR = 2.4 [95% CI: 1.2-4.4]; P = 0.008), liver (HR = 2.5 [95% CI: 1.2-5.1]; P = 0.01) or >1 metastatic compartment involvement (HR = 7.1 [95% CI: 1.0-48.4]; P = 0.047) at diagnosis. Neither HDC nor immunotherapy was associated with higher risk of CNS recurrence. Stable incidence of CNS relapse was reported over time. CONCLUSIONS: The risk of CNS recurrence is linked to both patient and disease characteristics, with neither impact of HDC nor immunotherapy. These findings support the current treatment strategy and do not justify a CNS prophylactic treatment.

High dose chemotherapy and autologous hematopoietic cell transplantation for Wilms tumor: a study of the European Society for Blood and Marrow Transplantation.

Survival for subgroups of patients with Wilms tumor (WT), such as those who suffer from relapse, is disappointing. Some patients' treatment plans include high-dose chemotherapy (HDT) with autologous hematopoietic cell transplantation (aHCT), although proof for its benefit is lacking. To increase the level of evidence regarding children with WT receiving aHCT as consolidation of first or second remission (after first relapse), we extracted relevant data from the European Blood and Marrow Transplantation Registry concerning 69 patients. Different HDT regimens were administered, mostly either melphalan-containing (n = 34) or thiotepa-containing (n = 14). For the whole population, 5-year overall survival (OS) and event-free survival (EFS) probabilities were 0.67 (±0.06) and 0.63 (±0.06), respectively (median observation time 7.8 years); for children transplanted in first remission, OS and EFS were 0.69 (±0.09) and 0.72 (±0.08). In univariate analysis, male gender and relapse in multiple sites were associated with lower OS probabilities. The use of a given pretransplant regimen (i.e. melphalan alone versus regimens with multiple drugs) did not seem to influence EFS/OS probability after aHCT, but significantly influenced platelet engraftment (more delayed with thiotepa). We here provide further data to improve the basis for future evidence-based clinical decision-making when using HDT and aHCT in relapsed/refractory WT.

Incidence and severity of crucial late effects after allogeneic HSCT for malignancy under the age of 3 years: TBI is what really matters.

Younger children are considered to be more vulnerable to late effects (LE), which prompted us to study LE in patients after haematopoietic stem cell transplantation (HSCT) for a haematological malignancy before the age of 3. In this multicentre EBMT study, cumulative incidence (CI) and severity of endocrine LE, central nervous system complications and secondary malignancies at 5, 10, 15 and 20 years of follow-up were assessed. Risk factors (RF) like gender, diagnosis, age at and year of HSCT, TBI- or chemo-conditioning and GVHD were analysed. CI of any LE was 0.30, 0.52, 0.66 and 0.72 at 5, 10, 15 and 20 years after HSCT, respectively. In 25% of the patients, LE were severe at a median follow-up of 10.4 years. In multivariate analysis, only TBI was a RF for having any LE and for thyroid dysfunction and growth disturbance. Female gender was a RF for delayed pubertal development. Some more insight could be gained by descriptive analysis regarding the role of TBI and GVHD on the severity of LE. Although only five selected LE have been studied and median follow-up is relatively short, the incidence and severity of these LE are considerable but not different from what has been found in older children and TBI is the main RF.

Hemopoietic stem cell transplantation in thalassemia: a report from the European Society for Blood and Bone Marrow Transplantation Hemoglobinopathy Registry, 2000-2010.

Allogeneic hemopoietic stem cell transplantation (HSCT) is the only method currently available to cure transfusion-dependent thalassemia major that has been widely used worldwide. To verify transplantation distribution, demography, activity, policies and outcomes inside the European Group for Blood and Marrow Transplantation (EBMT), we performed a retrospective non-interventional study, extracting data from the EBMT hemoglobinopathy prospective registry database. We included 1493 consecutive patients with thalassemia major transplanted between 1 January 2000 and 31 December 2010. In total, 1359 (91%) transplants were performed on patients <18 years old, 1061 were from a human leukocyte Ag-identical sibling donor. After a median observation time of 2 years, the 2-year overall survival (OS) and event-free survival (EFS; that is, thalassemia-free survival) were 88 ± 1% and 81 ± 1%, respectively. Transplantation from a human leukocyte Ag-identical sibling offered the best results, with OS and EFS of 91 ± 1% and 83 ± 1%, respectively. No significant differences in survival were reported between countries. The threshold age for optimal transplant outcomes was around 14 years, with an OS of 90-96% and an EFS of 83-93% when transplants were performed before this age. Allogeneic HSCT for thalassemia is a curative approach that is employed internationally and produces excellent results.

Persistence and reactivation of human adenoviruses in the gastrointestinal tract.

Reactivation of persistent human adenoviruses (HAdVs) is associated with high morbidity and mortality in paediatric haematopoietic stem cell transplant (HSCT) recipients. Although invasive HAdV infections mainly arise from the gastrointestinal (GI) tract, the specific sites of HAdV persistence are not well characterised. We prospectively screened biopsies from 143 non-HSCT paediatric patients undergoing GI endoscopy and monitored serial stool specimens from 148 paediatric HSCT recipients for the presence of HAdV by real-time PCR. Persistence of HAdV in the GI tract was identified in 31% of children, with the highest prevalence in the terminal ileum. In situ hybridisation and immunohistochemistry identified HAdV persistence in lymphoid cells of the lamina propria, whereas biopsies from five transplant recipients revealed high numbers of replicating HAdV in intestinal epithelial cells. The prevalence of HAdV species, the frequencies of persistence in the GI tract and reactivations post transplant indicated a correlation of intestinal HAdV shedding pre-transplant with high risk of invasive infection. HAdV persistence in the GI tract is a likely origin of infectious complications in immunocompromised children. Intestinal lymphocytes represent a reservoir for HAdV persistence and reactivation, whereas the intestinal epithelium is the main site of viral proliferation preceding dissemination. The findings have important implications for assessing the risk of life-threatening invasive HAdV infections.

Treosulfan-based conditioning regimens for allogeneic haematopoietic stem cell transplantation in children with non-malignant diseases.

An increasing number of children with non-malignant diseases can be cured by allogeneic haematopoietic stem cell transplantation (HSCT). Treosulfan (L-treitol-1,4-bis-methanesulfonate) is being used more frequently for conditioning, owing to its' lower toxicity profile compared with conventional myeloablative regimens. A retrospective analysis was performed of children registered in the EBMT database, who received treosulfan before HSCT between January 2005 and 2010, to identify possible dose-related toxicity and determine the incidence of engraftment, treatment-related mortality and overall survival (OS). Results from 316 transplants from 11 different countries are presented. Ninety-five (30%) were under 1 year of age at the time of transplant. OS was 83% and event-free survival was 76%; 3-year OS and event-free survival of infants below 1 year were 79% and 73%, respectively. No association was found with age at transplant, dose of treosulfan given, other agents used in combination with treosulfan, donor type, stem cell source, or second or subsequent transplant. In this report of the largest number of children to date receiving treosulfan for non-malignant diseases, treosulfan is shown to be a safe and effective agent even for those under 1 year of age at the time of transplant. Further prospective studies are needed using precisely defined protocols with pharmacokinetic monitoring and detailed chimerism analysis. In addition, long-term studies will be vital to determine long-term effects, for example, on fertility in comparison with other regimens.

Suppression of FOXO1 is responsible for a growth regulatory repressive transcriptional sub-signature of EWS-FLI1 in Ewing sarcoma.

The Ewing sarcoma (ES) EWS-FLI1 chimeric oncoprotein is a prototypic aberrant ETS transcription factor with activating and repressive regulatory functions. We report that EWS-FLI1-repressed promoters are enriched in forkhead box (FOX) recognition motifs, and identify FOXO1 as a EWS-FLI1-suppressed regulator orchestrating a major subset of EWS-FLI1-repressed genes. In addition to FOXO1 regulation by direct promoter binding of EWS-FLI1, its subcellular localization and activity is regulated by cyclin-dependent kinase 2- and AKT-mediated phosphorylation downstream of EWS-FLI1. Restoration of nuclear FOXO1 expression in ES cells impaired proliferation and significantly reduced clonogenicity. Gene-expression profiling revealed a significant overlap between EWS-FLI1-repressed and FOXO1-activated genes. As a proof of principle for a potential therapeutic application of our findings, the treatment of ES cell lines with methylseleninic acid (MSA) reactivated endogenous FOXO1 in the presence of EWS-FLI1 in a dose- and time-dependent manner and induced massive cell death dependent on FOXO1. In an orthotopic xenograft mouse model, MSA increased FOXO1 expression in the tumor paralleled by a significant decrease in ES tumor growth. FOXO1 reactivation by small molecules may therefore serve as a promising strategy for a future ES-specific therapy.

Exhaled nitric oxide and pulmonary complications after paediatric stem cell transplantation.

UNLABELLED: Pulmonary complications are major causes of morbidity and mortality after haematopoietic stem cell transplantation (HSCT). We hypothesise that elevated exhaled nitric oxide (FeNO) levels early after HSCT in children are predictive for pulmonary complications. The present prospective study included 30 children (age, 4-18 years) before HSCT. FeNO levels were evaluated 10 days before transplant, at day 0, day +28 and day +60 after HSCT. During the follow-up period until day +100, pulmonary complications and lung function were assessed. Before HSCT, the mean FeNO levels were comparable in children with or without post-transplant pulmonary complications. However, they differed at day 0 and day +28 with a mean of 7 (±1.95) and 13 (±3.44) ppb at day 0 and a mean of 13 (±3.44) and 14 (±3.57) ppb at day +28, respectively. CONCLUSION: Children with pulmonary complications after day +28 have higher mean FeNO levels 28 days after HSCT than children without later pulmonary complications. Therefore, FeNO could be an important diagnostic tool for hyperinflammatory response in bronchial epithelium after paediatric HSCT.

Non-atopic IgE and eosinophil cationic protein after allogeneic hematopoietic stem cell transplantation in children.

Allogeneic hematopoietic stem cell transplantation (HSCT) in childhood is associated with severe pulmonary complications, but the pathophysiologic mechanisms remain unclear. Our aim was to evaluate the association of total and specific IgE, eosinophil cationic protein (ECP) and eosinophilia in HSCT recipients with pulmonary complications. We prospectively measured total and specific serum IgE, eosinophils, and ECP before and 28, 100, and 180 days after HSCT. We included 30 children (age 2-17 years) undergoing HSCT. Nine patients had a history of previous atopy without being associated with pulmonary complications after HSCT until day +360. Specific IgE levels showed a decline after HSCT, associated with the absence of allergy symptoms, suggesting a reduction of atopy. Elevated total serum IgE levels occurred in seven patients on day +28 after HSCT. This elevation did not coincide with allergy symptoms. ECP showed no correlation with total allergy symptoms, eosinophilia, IgE levels, or pulmonary complications. There was a significant correlation (p = 0.0367) between ECP levels on day +28 and concurrent acute graft-versus-host disease (GvHD). Non-atopic serum ECP and IgE levels are elevated on day +28 after HSCT in children, with ECP showing a potential relation to acute GvHD.

Monitoring of adenovirus load in stool by real-time PCR permits early detection of impending invasive infection in patients after allogeneic stem cell transplantation.

Invasive adenovirus (AdV) infections are associated with high morbidity and mortality in allogeneic stem cell transplant recipients. We observed that molecular detection of the virus in stool specimens commonly precedes AdV viremia, suggesting that intestinal infections may represent a common source of virus dissemination. To address this notion, we have investigated 153 consecutive allogeneic transplantations in 138 pediatric patients by quantitative monitoring of AdV in stool specimens and peripheral blood by a pan-adenovirus real-time (RQ)-PCR approach. AdV was detectable in serial stool specimens in all cases of AdV viremia during the post-transplant course (P<0.0001). The incidence of AdV viremia in individuals with peak virus levels in stool specimens above 1 x 10E6 copies per gram (n=22) was 73% vs 0% in patients with AdV levels in stool specimens below this threshold (n=29; P<0.0001). Serial measurement of AdV levels in stool specimens by RQ-PCR permitted early diagnosis of impending invasive infection with a sensitivity and specificity of 100% (95% confidence interval (CI) 96-100%) and 83% (95% CI 67-92%), respectively. The median time span between detection of AdV loads in stool specimens above 1 x 10E6 copies per gram and first observation of viremia was 11 days (range 0-192). Quantitative monitoring of the AdV load in stool specimens therefore provides a rationale for early initiation of antiviral treatment with the aim of preventing progression to life-threatening invasive infection.

Induction death and treatment-related mortality in first remission of children with acute lymphoblastic leukemia: a population-based analysis of the Austrian Berlin-Frankfurt-Münster study group.

In the management of the childhood acute lymphoblastic leukemia (ALL), 5% of failures are due to induction death and treatment-related deaths in first complete remission. We retrospectively analyzed the incidence, pattern and causes of death and its risk factors for 896 children with ALL enrolled into five Austrian (A) Berlin-Frankfurt-Münster (BFM) trials between 1981 and 1999. The estimated 10-year cumulative incidence of death significantly decreased from 6+/-1% (n=16/268) in trials ALL-BFM-A 81 and ALL-A 84 to 2+/-1% (n=15/628) in trials ALL-BFM-A 86, 90 and 95 (P=0.006). A significant reduction of death was evident during induction therapy (2.2% in trials ALL-BFM-A 81 and ALL-A 84 and 0.2% in trials ALL-BFM-A 86, 90 and 95, P=0.001). Of 31 patients, 21 (68%) patients died from infectious and 10 (32%) from noninfectious complications. Treatment in trial ALL-BFM-A 81, infant age and female gender were independent predictors of an enhanced risk for death. Conclusively, we found a progressive reduction of death rates that may be explained by the increasing experience in specialized hemato-oncologic centers and improved supportive and intensive care. We also identified a distinct subset of patients who are especially prone to death and may need a special focus when receiving intense chemotherapy.

Chromosome 14 copy number-dependent IGH gene rearrangement patterns in high hyperdiploid childhood B-cell precursor ALL: implications for leukemia biology and minimal residual disease analysis.

Childhood B-cell precursor acute lymphoblastic leukemia (BCP ALL) is generally a clonal disease in which the number of IGH rearrangements per cell does not exceed the number of the IGH alleles on chromosome 14. Consequently, monoclonal high hyperdiploid (HeH) cases with a trisomy 14 can harbor three rearrangements, a pattern that otherwise may be misinterpreted to be oligoclonal. Oligoclonal IGH rearrangements, on the other hand, may be instable at relapse and should therefore not be used for minimal residual disease analysis. We thus investigated the association between IGH allele copy numbers and the IGH rearrangement patterns in 90 HeH BCP ALL with either two (13%) or three copies (87%) of chromosome 14. HeH cases (44%) had an oligoclonal IGH rearrangement pattern, but true oligoclonality--after correction for the respective copy number of IGH alleles--was only 16%. Monoclonal and oligoclonal HeH cases had predominantly V(H) to preexisting DJ(H) recombinations, a finding that contrasts with oligoclonal cases of other major genetic BCP ALL subgroups in which V(H) replacements prevail. We conclude that for the precise assessment and correct interpretation of clonality patterns in BCP ALL, the IGH allele copy number has to be taken into consideration.

Detection of human cytomegalovirus-specific T lymphocytes in human blood: comparison of two methods.

BACKGROUND: Human cytomegalovirus (HCMV) infection remains a major cause of morbidity and mortality in immunocompromised patients undergoing allogeneic stem cell transplantation (SCT). In the case of HCMV reactivation, the well-defined detection of virus-specific effector cells in patients might positively impact antiviral treatment. METHODS: We examined blood samples from healthy volunteers serologically typed for HCMV IgG. Based on multicolor flow cytometry analysis, we addressed HCMV-specific CD8(+) effector T lymphocytes using HCMV-specific tetramers for the respective major histocompatibility complex (MHC) class I type. As a second approach, we employed the cytokine secretion assay (CSA), which allows the indirect detection of target-specific CD4(+) and CD8(+) T cells via their interferon (IFN)-gamma secretion upon HCMV pp65 in vitro stimulation. RESULTS: We hypothesized the detection of HCMV-specific lymphocytes in >50% of healthy Caucasians that were IgG-seropositive for HCMV. In terms of specificity, both assays showed comparably good results (specificity 100%, confidence interval >95%). Regarding sensitivity, both assays met the zero hypothesis. However, with 45/52 (86.5%) the tetramer technology was superior to the CSA, which detected 34/52 (65.4%) based on CD8(+) T cells and 41/52 (78.8%) based on both CD4(+) and CD8(+) T cells. DISCUSSION: A good correlation was observed between both assays, although the tetramers addressed only CD8(+) HCMV-specific T cells, whereas IFN-gamma secretion was detected on all T-cell types. Disadvantages of the CSA are the time-consuming stimulation, the extensive cell washing steps and the fact that the target cells are detected indirectly. The analysis with tetramers is rapid and reliable but their general use is hampered because of the restriction to a few HLA types.

28 years of high-dose therapy and SCT for neuroblastoma in Europe: lessons from more than 4000 procedures.

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.

Risk factors for diabetes insipidus in langerhans cell histiocytosis.

BACKGROUND: Diabetes insipidus (DI) is the most frequent central nervous system (CNS)-related permanent consequence in Langerhans cell histiocytosis (LCH), which mostly requires life-long hormone replacement therapy. In an attempt to define the population at risk for DI, 1,741 patients with LCH registered on the trials DALHX 83 and DALHX 90, LCH I and LCH II were studied. RESULTS: Overall 212 of 1,741 patients (12%) was reported to have DI. In 102 of 1,741 patients (6%) DI was present at diagnosis of LCH. One thousand one hundred eighty three of 1,539 patients without DI at diagnosis had follow up information. One hundred ten of these (9%) later developed DI. The risk of developing DI was 20% at 15 years after diagnosis. Multisystem disease patients at diagnosis carried a 4.6-fold risk for DI compared to single system patients. Craniofacial lesions, in particular in the "ear," "eye," and oral region were associated with a significantly increased risk for DI (relative hazard rate, RHR 1.7), independent of the extent of disease. No influence of the duration of therapy could be determined, but the duration of initial disease activity (RHR 1.5) and the occurrence of reactivations (RHR 3.5) significantly increased the risk for DI. CONCLUSIONS: Patients with multisystem disease and craniofacial involvement at diagnosis, in particular of the "ear," "eye," and the oral region carry a significantly increased risk to develop DI during their course. This risk is augmented when the disease remains active for a longer period or reactivates.

Langerhans cell histiocytosis in neonates.

BACKGROUND: To study the incidence, clinical patterns, course, and outcome of neonatal Langerhans cell histiocytosis (LCH). PROCEDURE: Retrospective analysis of the data of the Austrian/German/Swiss/Netherlands LCH Study Group. The incidence of neonatal LCH was estimated with the data from the population-based German Childhood Cancer Registry. RESULTS: The estimated incidence of neonatal LCH (LCH diagnosed within 28 days after birth) in the population-based registry was 1-2/1,000,000. In 61/1,069 trial patients (6%), the first disease manifestations were observed in the neonatal period. However, in only 20 of them, the diagnosis was established within this period. There was a preponderance of multisystem (MS)-LCH 36/61 (59%). Cutaneous changes were the most common initial manifestation in both, single-system (SS)-LCH (92%), and MS-LCH (86%). In 72% of the MS-LCH patients, risk organs (ROs) were involved at diagnosis as well. The probability of survival at 5 years was 94% in SS-LCH and 57% in MS-LCH, which is significantly lower than in older age groups. CONCLUSIONS: In contrast to the available literature, neonatal LCH is characterized by a clear predominance of MS-LCH. Cutaneous changes are the most common initial manifestation in neonates with both SS-LCH and MS-LCH. Prompt evaluation of disease extent upon diagnosis is mandatory for risk-adapted treatment. The disease course is unpredictable upon diagnosis. Close monitoring for disease progression is mandatory if isolated cutaneous LCH is managed by the "wait and see" approach. Neonates with MS-LCH, especially those with RO involvement at diagnosis, have less favorable prognosis compared to infants and older children, and need systemic therapy.

High incidence and unique features of antigen receptor gene rearrangements in TEL-AML1-positive leukemias.

The t(12;21) translocation resulting in the TEL-AML1 gene fusion is found in 25% of childhood B-cell precursor (BCP) acute lymphoblastic leukemias (ALL). Since TEL-AML1 has been reported to induce cell cycle retardation and thus may influence somatic recombination, we analyzed 214 TEL-AML1-positive ALL by PCR for rearrangements of the immunoglobulin (Ig) and T-cell receptor (TCR) genes. As a control group, 174 childhood BCP ALL without a TEL-AML1 were used. The majority of TEL-AML1-positive leukemias had a higher number of Ig/TCR rearrangements than control ALL. They also had a more mature immunogenotype characterized by their high frequency of complete IGH, IGK-Kde, and TCRG rearrangements. While IGK-Kde and TCRG were more frequently rearranged on both alleles at higher age, IGH and TCRD rearrangements decreased in their incidence along with a decrease in biallelic IGH rearrangements. This suggests that the recombination process continues in these leukemias leading to ongoing rearrangements and possibly also deletions of antigen receptor genes. We here provide first evidence that somatic recombination of antigen receptor genes is affected by the TEL-AML1 fusion, and that further age-related differences are probably caused by the longer latency period of the prenatally initiated TEL-AML1-positive leukemias in older children.

Quality assessment of genetic markers used for therapy stratification.

PURPOSE: Therapy stratification based on genetic markers is becoming increasingly important, which makes commitment to the highest possible reliability of the involved markers mandatory. In neuroblastic tumors, amplification of the MYCN gene is an unequivocal marker that indicates aggressive tumor behavior and is consequently used for therapy stratification. To guarantee reliable and standardized quality of genetic features, a quality-assessment study was initiated by the European Neuroblastoma Quality Assessment (ENQUA; connected to International Society of Pediatric Oncology) Group. MATERIALS AND METHODS: One hundred thirty-seven coded specimens from 17 tumors were analyzed in 11 European national/regional reference laboratories using molecular techniques, in situ hybridization, and flow and image cytometry. Tumor samples with divergent results were re-evaluated. RESULTS: Three hundred fifty-two investigations were performed, which resulted in 23 divergent findings, 17 of which were judged as errors after re-evaluation. MYCN analyses determined by Southern blot and in situ hybridization led to 3.7% and 4% of errors, respectively. Tumor cell content was not indicated in 32% of the samples, and 11% of seemingly correct MYCN results were based on the investigation of normal cells (eg, Schwann cells). Thirty-eight investigations were considered nonassessable. CONCLUSION: This study demonstrated the importance of revealing the difficulties and limitations for each technique and problems in interpreting results, which are crucial for therapeutic decisions. Moreover, it led to the formulation of guidelines that are applicable to all kinds of tumors and that contain the standardization of techniques, including the exact determination of the tumor cell content. Finally, the group has developed a common terminology for molecular-genetic results.

Prognostic impact of chromosomal aberrations in Ewing tumours.

Although greater than 50% of Ewing tumours contain non-random cytogenetic aberrations in addition to the pathognomonic 22q12 rearrangements, little is known about their prognostic significance. To address this question, tumour samples from 134 Ewing tumour patients were analysed using a combination of classical cytogenetics, comparative genomic and fluorescence in situ hybridisation. The evaluation of the compiled data revealed that gain of chromosome 8 occurred in 52% of Ewing tumours but was not a predictive factor for outcome. Gain of 1q was associated with adverse overall survival and event-free survival in all patients, irrespective of whether the tumour was localised or disseminated (overall survival: P=0.002 and P=0.029; event-free survival: P=0.018 and P=0.010). Loss of 16q was a significant predictive factor for adverse overall survival in all patients (P=0.008) and was associated with disseminated disease at diagnosis (P=0.039). Gain of chromosome 12 was associated with adverse event-free survival (P=0.009) in patients with localised disease. These results indicate that in addition to a 22q12 rearrangement confirmation in Ewing tumours it is important to assess the copy number of 1q and 16q to identify patients with a higher probability of adverse outcome.