Segmental chromosomal alterations have prognostic impact in neuroblastoma: a report from the INRG project.

BACKGROUND: In the INRG dataset, the hypothesis that any segmental chromosomal alteration might be of prognostic impact in neuroblastoma without MYCN amplification (MNA) was tested. METHODS: The presence of any segmental chromosomal alteration (chromosome 1p deletion, 11q deletion and/or chromosome 17q gain) defined a segmental genomic profile. Only tumours with a confirmed unaltered status for all three chromosome arms were considered as having no segmental chromosomal alterations. RESULTS: Among the 8800 patients in the INRG database, a genomic type could be attributed for 505 patients without MNA: 397 cases had a segmental genomic type, whereas 108 cases had an absence of any segmental alteration. A segmental genomic type was more frequent in patients >18 months and in stage 4 disease (P<0.0001). In univariate analysis, 11q deletion, 17q gain and a segmental genomic type were associated with a poorer event-free survival (EFS) (P<0.0001, P=0.0002 and P<0.0001, respectively). In multivariate analysis modelling EFS, the parameters age, stage and a segmental genomic type were retained in the model, whereas the individual genetic markers were not (P<0.0001 and RR=2.56; P=0.0002 and RR=1.8; P=0.01 and RR=1.7, respectively). CONCLUSION: A segmental genomic profile, rather than the single genetic markers, adds prognostic information to the clinical markers age and stage in neuroblastoma patients without MNA, underlining the importance of pangenomic studies.

International consensus for neuroblastoma molecular diagnostics: report from the International Neuroblastoma Risk Group (INRG) Biology Committee.

Neuroblastoma serves as a paradigm for utilising tumour genomic data for determining patient prognosis and treatment allocation. However, before the establishment of the International Neuroblastoma Risk Group (INRG) Task Force in 2004, international consensus on markers, methodology, and data interpretation did not exist, compromising the reliability of decisive genetic markers and inhibiting translational research efforts. The objectives of the INRG Biology Committee were to identify highly prognostic genetic aberrations to be included in the new INRG risk classification schema and to develop precise definitions, decisive biomarkers, and technique standardisation. The review of the INRG database (n=8800 patients) by the INRG Task Force finally enabled the identification of the most significant neuroblastoma biomarkers. In addition, the Biology Committee compared the standard operating procedures of different cooperative groups to arrive at international consensus for methodology, nomenclature, and future directions. Consensus was reached to include MYCN status, 11q23 allelic status, and ploidy in the INRG classification system on the basis of an evidence-based review of the INRG database. Standardised operating procedures for analysing these genetic factors were adopted, and criteria for proper nomenclature were developed. Neuroblastoma treatment planning is highly dependant on tumour cell genomic features, and it is likely that a comprehensive panel of DNA-based biomarkers will be used in future risk assignment algorithms applying genome-wide techniques. Consensus on methodology and interpretation is essential for uniform INRG classification and will greatly facilitate international and cooperative clinical and translational research studies.

Expression and sequence analysis of candidates for the 1p36.31 tumor suppressor gene deleted in neuroblastomas.

Neuroblastomas are characterized by 1p deletions, suggesting that a tumor suppressor gene (TSG) resides in this region. We have mapped the smallest region of deletion (SRD) to a 2 Mb region of 1p36.31 using microsatellite and single nucleotide polymorphisms. We have identified 23 genes in this region, and we have analysed these genes for mutations and RNA expression patterns to identify candidate TSGs. We sequenced the coding exons of these genes in 30 neuroblastoma cell lines. Although rare mutations were found in 10 of the 23 genes, none showed a pattern of genetic change consistent with homozygous inactivation. We examined the expression of these 23 genes in 20 neuroblastoma cell lines, and most showed readily detectable expression, and no correlation with 1p deletion. However, 7 genes showed uniformly low expression in the lines, and 2 genes (CHD5, RNF207) had virtually absent expression, consistent with the expected pattern for a TSG. Our mutation and expression analysis in neuroblastoma cell lines, combined with expression analysis in normal tissues, putative function and prior implication in neuroblastoma pathogenesis, suggests that the most promising TSG deleted from the 1p36 SRD is CHD5, but TNFRSF25, CAMTA1 and AJAP1 are also viable candidates.

Expression of Apo-3 and Apo-3L in primitive neuroectodermal tumours of the central and peripheral nervous system.

Deregulation of apoptosis has been implicated in the pathogenesis, spontaneous regression and treatment resistance of neuroblastoma. A newly recognised member of the tumour necrosis factor (TNF)-family of death receptors known as Apo-3 has been mapped to human chromosome 1p36.3, a region commonly deleted in aggressive neuroblastoma. Based on its localisation and function, Apo-3 is a candidate for the putative neuroblastoma tumour suppressor gene. Therefore we analysed mRNA expression of the Apo-3 receptor/ligand (Apo-3/Apo-3L) system in a representative panel of 18 neuroblastoma cell lines, 41 primary neuroblastoma and 13 ganglioneuromas/ganglioneuroblastomas by semi-quantitative RT-PCR. We compared the level of expression with the well-established prognostic factors age, stage, histology, MYCN-amplification and TrkA expression, as well as outcome. For comparison, we studied Apo-3/Apo-3L expression in 27 central nervous system (CNS) primitive neuroectodermal tumours/medulloblastomas (PNET/medulloblastoma) and in six normal brain samples. Neuroblastoma cell lines with 1p deletion and MYCN-amplification expressed significantly lower levels of Apo-3 (P=0.009 and P=0.03, respectively) compared with neuroblastoma cell lines without 1p deletion or MYCN-amplification. The mean expression level of Apo-3L was significantly higher in ganglioneuromas/ganglioneuroblastomas compared with neuroblastomas (P=0.001) and in normal brain compared with PNET/medulloblastoma (P<0.0001). Expression of Apo-3L was significantly associated with survival in neuroblastomas (P<0.049) and in PNET/medulloblastomas (P=0.01). Expression of Apo-3 was significantly associated with survival in PNET/medulloblastomas (P=0.03). Thus, the Apo-3 receptor/ligand system might be involved in the regulation of apoptosis in neuroblastomas and PNET.

Histopathology (International Neuroblastoma Pathology Classification) and MYCN status in patients with peripheral neuroblastic tumors: a report from the Children's Cancer Group.

BACKGROUND: The International Neuroblastoma Pathology Classification (International Classification), which was established in 1999, is significant prognostically and is relevant biologically for the evaluation and analysis of patients with neuroblastic tumors (NTs). MYCN amplification is a known molecular marker for aggressive progression of NTs. These have been used together as important prognostic factors to define risk groups for patient stratification and protocol assignment. METHODS: A total of 628 NTs (535 neuroblastomas [NBs]); 21 ganglioneuroblastoma, intermixed [GNBi]; 9 ganglioneuromas [GN]; and 63 ganglioneuroblastoma, nodular [GNBn]) from the Children's Cancer Group studies were evaluated histologically (favorable histology [FH] tumors vs. unfavorable histology [UH] tumors) according to the International Classification and were tested molecularly for MYCN status (amplified vs. nonamplified). Four tumor subsets (FH-nonamplified, FH-amplified, UH-nonamplified, and UH-amplified) were defined by histopathology and MYCN status, and their prognostic effects were analyzed. Detailed analysis between morphologic indicators (grade of neuroblastic differentiation and mitosis-karyorrhexis index [MKI]) and MYCN status was done by using tumors in the NB category. RESULTS: There were 339 FH-nonamplified tumors (5-year event free survival [EFS], 92.1%); 8 FH-amplified tumors (EFS, 37.5%); 172 UH-nonamplified tumors (EFS, 40.9%); and 109 UH-amplified tumors (EFS, 15.0%). The prognostic effects on patients with tumors in the four subsets were independent from the factors of patient age and disease stage (P < 0.0001). MYCN amplification was seen almost exclusively in tumors of the NB category, and no patients with tumors in either the GNBi category or in the GN category and only two patients with tumors in the GNBn category had amplified MYCN. Among the patients with tumors in the NB category, patients with FH-nonamplified tumors (309 patients) had an excellent prognosis, and patients with UH-amplified tumors (107 patients) had the poorest clinical outcome in any age group. The prognosis for children with UH-nonamplified tumors (111 patients) was poor when they were diagnosed at age > 1.5 years. It was also noted that patients with UH-amplified tumors (median age, 2.14 years) were diagnosed at a significantly younger age compared with the patients with UH-nonamplified tumors (median age, 3.55 years). Histologically, MYCN-amplified tumors lacked neuroblastic differentiation regardless of the age of patients. MYCN amplification also was linked generally to increased mitotic and karyorrhectic activities. However, MKI classes in patients with MYCN-amplified tumors varied significantly, depending on the age at diagnosis, and younger patients had higher MKI classes. CONCLUSIONS: The combination of histopathologic evaluation and MYCN status distinguishes four clinical and biologic tumor subsets in patients with NTs. MYCN amplification seems to be the powerful driving force for preventing cellular differentiation regardless of patient age and for increasing mitotic and karyorrhectic activities in an age dependent manner.

[Expression of the neurotrophin-receptor TrkB predicts outcome in nephroblastomas: results of a pilot-study]. / Die Expression des Neurotrophin-Rezeptors TrkB beeinflusst die Prognose von Nephroblastomen: Ergebnisse einer Pilotstudie.

BACKGROUND: The neurotrophin-receptor TrkB plays an important role in pathogenesis, biology and prognosis of neuroblastoma. Expression of TrkB on aggressive neuroblastomas leads to proliferation and survival of the tumor cells and is associated with an unfavorable prognosis. It is now known that Trk receptors are also expressed in extraneural tissues including the kidney. PATIENTS AND METHODS: To study the role of the neurotrophin-receptor TrkB in nephroblastoma/Wilms' Tumor (WT), we determined TrkB mRNA expression by semiquantitative duplex RT-PCR in 39 primary WT. Comparison of mRNA expression levels with clinical variables was performed using Cox regression analysis. RESULTS: The 5-year overall survival was significantly worse for patients with tumors expressing high levels of a functional TrkB-receptor (TrkBfull) in comparison to patients with low levels of TrkBfull (70 % versus 100 %, p=0.005). Conversely, children with tumors expressing high mRNA levels of a functionally inactive truncated TrkB receptor (TrkBtrunc) had a significantly higher 5-year overall survival rate in comparison to patients with low levels of TrkBtrunc (100 % versus 68 %, p=0.003). The hazard ratios for TrkBfull and TrkBtrunc remained significant after adjusting for tumor stage. All WT with high levels of TrkB also expressed the ligand brain-derived neurotrophic factor (BDNF). CONCLUSIONS: Full-length and truncated TrkB appear to be important prognostic factors in WT. Their expression should be assessed prospectively in a larger panel of WT and may have a future role in patient assignment to risk-based treatment strategies. TrkB signaling may be reduced in WT with favorable outcome due to low numbers of TrkB receptors or a competitive effect of functionally inactive TrkBtrunc.

MYC messenger RNA expression predicts survival outcome in childhood primitive neuroectodermal tumor/medulloblastoma.

PURPOSE AND EXPERIMENTAL DESIGN: Cerebellar primitive neuroectodermal tumors/medulloblastomas (PNET/MB) are the most common malignant brain tumors in childhood. To identify PNET/MB biological prognostic factors that define a patient group with a sufficiently good prognosis to permit a reduction in treatment intensity, we determined the expression levels of MYC mRNA in fresh frozen tumor samples from 26 PNET/MB patients using semiquantitative reverse transcription-PCR. RESULTS: MYC mRNA expression levels in primary PNET/MB showed a wide range with a 22-fold difference between the highest and lowest values and did not correlate with MYC gene amplification. MYC mRNA expression was an independent significant prognostic factor for progression-free survival outcome and was more predictive than standard clinical factors. The combination of low MYC mRNA expression and high TrkC mRNA expression identified a good outcome group of PNET/MB patients (n = 7) with 100% progression-free survival after a median follow-up time of 55 months (range, 15-91 months). Three of these seven good outcome patients survived without radiotherapy. CONCLUSIONS: Low MYC mRNA expression is a powerful independent predictor of favorable clinical outcome in PNET/MB. Assessment of MYC mRNA levels is feasible and may be incorporated in prospective PNET/MB clinical trials to aid in treatment planning for patients with PNET/MB on confirmation of our results in larger studies.

TrkA signal transduction pathways in neuroblastoma.

BACKGROUND: Favorable neuroblastomas frequently express high levels of the TrkA receptor, and these tumors have a propensity to either differentiate or regress, but the mechanisms responsible for these two fates are unclear. PROCEDURE: To study TrkA signal transduction in neuroblastoma (nb), we stably expressed wild-type TrkA and five TrkA mutants in the human nb cell line SH-SY5Y. Resulting single cell clones were characterized by TrkA mRNA and protein expression and by autophosphorylation of the receptor. RESULTS: Introduction of TrkA restored nerve growth factor (NGF) responsiveness of SH-SY5Y cells, demonstrated by morphological differentiation and induction of immediate-early genes. TrkA overexpression leads to growth inhibition in the absence of NGF, whereas NGF treatment results in increased proliferation. CONCLUSIONS: Analysis of downstream signaling elements in mutated TrkA receptors indicates that NGF-induced differentiation is dependent on TrkA kinase activity, but several redundant pathways seem to be used farther downstream. This suggests differences from TrkA pathways identified in PC12 cells.

Biological aspects of neuroblastomas identified by mass screening in Quebec.

BACKGROUND: Neuroblastoma has several characteristics that suggest that preclinical diagnosis might improve outcome. Therefore, the Quebec Neuroblastoma Screening Project was undertaken from 1989 to 1994 to examine infants at 3 weeks and 6 months by measuring urinary catecholamine metabolites. PROCEDURE: Over the 5-yr period, 45 tumors were detected by screening, 20 were identified clinically prior to the third week, and 64 were identified clinically at a later time. We analyzed available tumors for Shimada histopathology, tumor ploidy, MYCN copy number and serum ferritin. RESULTS: Of the tumors detected by screening, only 2 of 45 tested had unfavorable histology, 2 of 45 had diploid or tetraploid DNA content, 0 of 43 had MYCN amplification, and 4 of 44 had elevated serum ferritin. All of these patients are alive and well. The 20 patients detected prior to the 3-week screen had similar biological characteristics. In contrast, of the patients detected clinically after 3 weeks of age, 19 of 51 testedhad unfavorable histology, 25 of 66 had diploid or tetraploid tumors, 12 of 56 had MYCN amplification, and 14 of 54 had elevated ferritin. CONCLUSIONS: The difference between the screened and clinically detected cases was highly significant for each biological variable. Preliminary data on other biological variables, such as neurotrophin expression and allelic loss on 1 p in these patients are consistent with the above findings. These data suggest that mass screening for neuroblastoma at or before 6 months of age detects almost exclusively tumors that have favorable biological characteristics, many of which might have regressed spontaneously. Thus, continued mass screening for neuroblastoma at 6 months is unlikely to accomplish its intended goal, and should probably be discontinued.

Effect of CEP-751 (KT-6587) on neuroblastoma xenografts expressing TrkB.

BACKGROUND: The compound CEP-751 (KT-6587), a potent and selective inhibitor of the Trk family of tyrosine kinases, has been shown to inhibit the growth of human neuroblastoma (NB) xenografts in nude mice [1]. PROCEDURE: To address its mechanism of action, we studied SY5Y, a human NB cell line with no detectable Trk expression, and two subclones transfected with TrkB. The transfected clones, SY5Y (G8) and SY5Y (G12), expressed moderate and high levels, respectively, of TrkB mRNA and protein. These TrkB-expressing subclones and the parental line were then grown as xenografts in nude mice, and CEP-751 was used to inhibit TrkB tyrosine kinase activity in these xenografts. Animals were treated twice a day with CEP-751 (21 mg/kg), or with the carrier vehicle as a control. TrkB expression in the resultant tumors was examined by quantitative RT-PCR. The effect of CEP-751 on TrkB activation by BDNF was examined in G12 cells in culture by immunoprecipitation with antipan Trk antiserum, followed by Western blot analysis using antiphosphotyrosine antibodies. To determine if CEP-751 was causing apoptosis, the TUNEL assay was used. RESULTS: CEP-751 had little effect on the growth of SY5Y tumors, but did slow the growth rate of the C8 and G12 tumors. The daily growth rate of the treated tumors was 0.16, 0.13, and 0.10 cm3, respectively, for the SY5Y, G8, and G12 tumors. RT PCR analysis confirmed the expression of TrkB in G8 and G12, but not in SY5Y tumors. Activation of TrkB by BDNF in G12 cells was inhibited by CEP-751 in a dose dependent fashion. The treated tumors showed marked evidence of apoptosis. CONCLUSIONS: These data suggest that the effect of CEP-751 is due, at least in part, to its inhibition of TrkB kinase, and that CEP-751 may become a useful therapeutic tool for the treatment of aggressive neuroblastomas, which often express TrkB.

Allelic deletion at chromosome bands 11q14-23 is common in neuroblastoma.

BACKGROUND: Neuroblastoma tumorigenesis may involve the differential inactivation of multiple tumor suppressor genes. Recent data have suggested that a neuroblastoma suppressor gene may be located on the long arm of chromosome 11 (11q). PROCEDURE: We therefore analyzed 295 primary neuroblastomas from a representative group of patients for loss of heterozygosity (LOH) at 25 polymorphic markers spanning 11q. RESULTS: LOH was observed in 129 primary neuroblastomas (44%), and a common region of LOH mapped to 11q14-23. No correlation was found between 11q LOH and adverse prognostic variables, but a strong inverse relationship between 11q LOH and MYCN amplification (P < 0.001) was observed. There was no difference in overall survival when patients were stratified by 11q LOH status. However, 11q LOH was associated with a decreased overall survival probability when patients whose tumors had a single copy of MYCN were analyzed separately (P = 0.008). CONCLUSION: These data support the hypothesis that a tumor suppressor gene mapping within 11q14-23 is frequently inactivated during the malignant evolution of neuroblastoma.

Loss of heterozygosity for chromosome 14q in neuroblastoma.

BACKGROUND: Neuroblastoma is a genetically heterogeneous disease, with subsets of tumors demonstrating rearrangements of several genomic regions. Preliminary studies from several groups have identified loss of heterozygosity (LOH) for the long arm of chromosome 14 (14q) in 20-25% of primary neuroblastomas. PROCEDURE: To determine precisely the frequency and extent of 14q deletions, we performed LOH analysis for a large series of primary neuroblastomas using a panel of 11 highly polymorphic markers. RESULTS: LOH was detected in 83 of 372 tumors (22%). Although the majority of tumors with allelic loss demonstrated allelic loss for all informative markers, 13 cases showed LOH for only a portion of 14q. A single consensus region of deletion, which was shared by all tumors with 14q LOH, was defined within 14q23-q32 between D14S588 and the 14q telomere. Allelic loss for 14q was strongly correlated with the presence of 11q LOH (P < 0.001 ) and inversely correlated with MYCN amplification (P= 0.04). CONCLUSIONS: LOH for 14q was evident in all clinical risk groups, indicating that this abnormality may be a universal feature of neuroblastoma tumor development. These findings suggest that a tumor suppressor gene involved in the initiation or progression of neuroblastoma is located within distal 14q.

Comprehensive analysis of chromosome 1p deletions in neuroblastoma.

BACKGROUND: Chromosome 1p deletions are common in advanced neuroblastomas, but the biological and clinical implications of this clonal rearrangement remain controversial. Previous studies of chromosome 1p loss of heterozygosity (LOH) have been limited by analyses of relatively small number of tumors derived from heterogeneously assessed and treated patient populations. Therefore, a strictly representative cohort of 288 Children's Cancer Group neuroblastoma patients treated on the most recent phase III therapeutic trials was identified. PROCEDURE: Primary tumors from these patients were analyzed for LOH at precisely mapped and highly informative 1p polymorphic loci located from 1p32 to 1p36.3 by multiplex PCR. RESULTS: Ninety-three primary tumor specimens (32%) had LOH at multiple 1p36 marker loci. All 1p deletions overlapped the previously determined smallest region of overlap (SRO). One tumor had a small terminal deletion completely within 1p36.3, allowing for further refinement of the 1p36 SRO. We found no evidence to support an additional, nonoverlapping region of LOH within 1p32-36. We confirmed the strong correlation of 1p36 LOH with MYCN amplification (P < 0.001), advanced disease stage (P < 0.001), and decreased both 3-year event-free survival and overall survival probabilities (P< 0.001). When stratified for MYCN amplification status or entered into a multivariate analysis, 1p36 LOH remained predictive for decreased event-free survival, but not overall survival probability. CONCLUSIONS: These data support the hypothesis that inactivation of a tumor suppressor gene within 1p36.3 is associated with an increased risk for disease relapse.

Detailed molecular analysis of 1p36 in neuroblastoma.

BACKGROUND: Several lines of evidence es tablish that chromosome band 1p36 is frequently deleted in neuroblastoma primary tumors and cell lines, suggesting that a tumor suppressor gene within this region is involved in the development of this tumor. PROCEDURE: We analyzed the status of 1p36 in primary neuroblastomas and cell lines to define the region of consistent rearrangement. RESULTS: Loss of heterozygosity (LOH) studies of primary neuro blastomas identified allelic loss in 135 of 503 tumors (27%), with the smallest region of overlap (SRO) defined distal to D15214 (1p36.3). No homozygous deletions were detected at 120 loci mapping to 1p36.1-p36.3 in a panel of 46 neuroblastoma cell lines. A recently identified patient with neuroblastoma was found to have a constitutional deletion within 1p36.2-p36.3, and this deletion, when combined with the LOH results, defined a smaller SRO of one megabase within 1p36.3. We constructed a comprehensive integrated map of chromosome 1 containing 11,000 markers and large-insert clones, a high-resolution radiation hybrid (RH) map of 1p36, and a P1-artificial chromosome (PAC) contig spanning the SRO, to further characterize the region of interest. Over 768 kb (75%) of the SRO has been sequenced to completion. Further analysis of distal 1p identified 113 transcripts localizing to 1p36, 21 of which were mapped within the SRO. CONCLUSION: This analysis will identify suitable positional candidate transcripts for mutational screening and subsequent identification of the 1p36.3 neuroblastoma suppressor gene.

Analysis of genomic imprinting at 1p35-36 in neuroblastoma.

BACKGROUND: Deletion of the distal short arm of chromosome 1 occurs in 25-35% of primary neuroblastomas, and a putative tumor suppressor gene has been mapped to a consensus region of deletion at 1p36.2-36.3. Indirect evidence suggests the presence of an imprinted neuroblastoma suppressor gene within this region, as well as an additional nonimprinted, proximal suppressor gene, inactivation of which correlates with MYCN amplification. PROCEDURE: To test this hypothesis, we performed 1p loss of heterozygosity (LOH) studies on a series of neuroblastomas for which parental DNA had been collected. PCR-formatted polymorphic markers were used to determine the size of the 1p deletion and the parental origin of the deleted 1p homologue. RESULTS: Twenty-six neuroblastomas with 1p LOH were evaluated. Twenty-four had MYCN amplification, and of these, 15 demonstrated loss of the paternally inherited 1p. Two neuroblastomas with a single copy of MYCN were evaluated and both had deletion of the paternally inherited 1p, with one case exhibiting a small terminal deletion. In addition, we have reviewed 49 previously reported neuroblastomas where 1p LOH data and the parental origin of the deleted lp homologue were available. CONCLUSIONS: Analyzed together, these 75 neuroblastomas demonstrate random deletion of parental 1p homologues (P = 0.30). Further, tumors with smaller deletions (breakpoints distal to D1S201 or D1S7) showed a random loss of the parental 1p homologues (P = 0.59), contrary to the expected preferential maternal 1p deletion if an imprinted suppressor gene mapped to this region. However, 19 tumors with 1p LOH and single copy MYCN had deletion of the maternal 1p homologue preferentially (P = 0.02), which does not exclude the possibility that loss of an imprinted suppressor gene plays a role in this subset.

Association among EPHB2, TrkA, and MYCN expression in low-stage neuroblastomas.

BACKGROUND: The EPH family is the largest subfamily of receptor protein-tyrosine kinases, consisting of EPHA and EPHB subgroups. Ligands of EPH family receptors are called ephrins, which include ephrin-A and ephrin-B subgroups. We recently found that transcripts encoding the EPHB subgroup (EPHB) and the ephrin-B subgroup (EFNB) were expressed together in neuroblastoma (NB) cell lines. PROCEDURE: In this study, we examined the expression of EPHB and EFNB transcripts in 24 NB specimens representing all clinical stages. We found that several EPHB and EFNB transcripts were expressed together in all NBs examined. RESULTS: Among the transcripts examined, EPHB6 expression was most significantly associated with low stage tumors (stages 1, 2, and 4S; P = 0.0048). TrkA expression was significantly correlated with EPHB6, EFNB2, and EFNB3 expression (P < 0.01 in each case). Taken together, these data indicate that the expression of EPHB6, EFNB2, and EFNB3 may serve as prognostic indicators of favorable NBs. In the low-stage NBs without MYCN amplification, EPHB2 expression was correlated both with MYCN expression and with TrkA expression (P < 0.01 in each case). Moreover, MYCN expression was correlated with TrkA expression (P < 0.01) in the low-stage NBs. CONCLUSIONS: This observation points to the possibility that MYCN expression might contribute to favorable outcome of low-stage NBs.

Detection of MYCN gene amplification in neuroblastoma by fluorescence in situ hybridization: a pediatric oncology group study.

To assess the utility of fluorescence in situ hybridization (FISH) for analysis of MYCN gene amplification in neuroblastoma, we compared this assay with Southern blot analysis using tumor specimens collected from 232 patients with presenting characteristics typical of this disease. The FISH technique identified MYCN amplification in 47 cases, compared with 39 by Southern blotting, thus increasing the total number of positive cases by 21%. The major cause of discordancy was a low fraction of tumor cells (< or =30% replacement) in clinical specimens, which prevented an accurate estimate of MYCN copy number by Southern blotting. With FISH, by contrast, it was possible to analyze multiple interphase nuclei of tumor cells, regardless of the proportion of normal peripheral blood, bone marrow, or stromal cells in clinical samples. Thus, FISH could be performed accurately with very small numbers of tumor cells from touch preparations of needle biopsies. Moreover, this procedure allowed us to discern the heterogeneous pattern of MYCN amplification that is characteristic of neuroblastoma. We conclude that FISH improves the detection of MYCN gene amplification in childhood neuroblastomas in a clinical setting, thus facilitating therapeutic decisions based on the presence or absence of this prognostically important biologic marker.

Opsoclonus-myoclonus-ataxia syndrome in neuroblastoma: clinical outcome and antineuronal antibodies-a report from the Children's Cancer Group Study.

BACKGROUND: Opsoclonus-myoclonus-ataxia (OMA) is a paraneoplastic neurologic syndrome affecting 2-3% of children with neuroblastoma. Although children with OMA and neuroblastoma may have higher survival, many experience a significant amount of late neurologic impairment, which may be immunologically mediated. The aim of this study was to compare the outcome of neuroblastoma patients with and without OMA, relating to prognostic factors, treatment, and the presence or absence of anti-neuronal antibodies. PROCEDURE: Questionnaires were mailed out requesting information on the current neurologic status of patients who submitted sera at diagnosis to the Children's Cancer Group serum bank from 1980 to 1994. Information was requested on clinical and biological patient characteristics as well as clinical aspects of the patients identified as having OMA syndrome, including presentation and treatment for OMA, late sequelae of OMA, the presence or absence of antineuronal antibodies, and survival. Sera from 16 of the OMA patients and 48 case-controls with neuroblastoma were assayed for anti-neuronal antibodies. RESULTS: Of the 675 responses received, 21 patients had OMA. Ninety percent of OMA patients presented with non-metastatic disease, vs. 35% of non-OMA patients. Estimated 3-year survival for the OMA patients with nonmetastatic disease (stage I, II, III) greater than 1 year of age was 100% vs. 77% for similar non-OMA patients (P = 0.0222). At follow-up, 14/19 evaluable OMA patients displayed some form of developmental or neurologic abnormality. There was no significant correlation of late sequelae with antineuronal antibodies, age, time between OMA symptoms and diagnosis, or treatment given for tumor or OMA. There was a significant correlation of late sequelae with lower stage disease (I and II) compared to more advanced disease (III and IV). CONCLUSIONS: Patients with OMA and neuroblastoma have excellent survival but a high risk of neurologic sequelae. Favorable disease stage correlates with a higher risk for development of neurologic sequelae. The role of anti-neuronal antibodies in late sequelae of OMA needs further clarification.

Resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in neuroblastoma cells correlates with a loss of caspase-8 expression.

Disruption of apoptotic pathways may be involved in tumor formation, regression, and treatment resistance of neuroblastoma (NB). Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis in cancer cell lines, whereas normal cells are not sensitive to TRAIL-mediated apoptosis. In this study we analyzed the expression and function of TRAIL and its agonistic and antagonistic receptors as well as expression of cellular FLICE-like inhibitory protein and caspase-2, -3, -8, -9, and -10 in 18 NB cell lines. Semiquantitative RT-PCR revealed that TRAIL-R2 and TRAIL-R3 are the main TRAIL-receptors used by NB cells. Sensitivity to TRAIL-induced apoptosis did not correlate with mRNA expression of TRAIL receptors or cellular FLICE-like inhibitory protein. Surprisingly, caspase-8 and caspase-10 mRNA expression was detected in only 5 of 18 NB cell lines. Interestingly, only these five NB cell lines were susceptible to TRAIL-induced apoptosis in a time- and dose-dependent manner. Treatment with 5-aza-2'-deoxycytidine restored mRNA and protein expression of caspase-8 and TRAIL sensitivity of resistant cell lines, suggesting that gene methylation is involved in caspase inactivation. The TRAIL system seems to be functional in NB cells expressing caspase-8 and/or caspase-10. Because many cytotoxic drugs induce caspase-dependent apoptosis, failure to express caspase-8 and/or caspase-10 might be an important mechanism of resistance to chemotherapy in NB.

Expression of the neurotrophin receptor TrkB is associated with unfavorable outcome in Wilms' tumor.

PURPOSE: Neurotrophins and their receptors regulate the proliferation, differentiation, and death of neuronal cells, and they have been implicated in the pathogenesis and prognosis of neuroblastomas and medulloblastomas. Tyrosine kinase (Trk) receptors also are expressed in extraneural tissues. PATIENTS AND METHODS: To study the role of neurotrophin receptors and ligands in Wilms' tumor (WT), we determined their expression by semiquantitative duplex reverse transcriptase polymerase chain reaction in 39 patients with primary WT. Comparison of mRNA expression levels with clinical variables was performed by use of Cox regression analysis. RESULTS: Children with WT that expressed high levels of full-length TrkB mRNA (TrkBfull) had a significantly greater risk of death than children whose tumors had little or no TrkBfull expression (hazard ratio, 9.7; P =.02). The 5-year relapse-free survival was 100% versus 65% for patients with low versus high tumor expression of TrkBfull (P <.003). Conversely, children with tumors that expressed high mRNA levels of a functionally inactive truncated TrkB receptor (TrkBtrunc) had a greater chance of survival than children with low levels of TrkBtrunc (hazard ratio, 0.08; P =.005). The 5-year relapse-free survival was 95% versus 68% for patients with high versus low levels of TrkBtrunc (P =.01). The hazard ratios for TrkBfull and TrkBtrunc remained significant after they were adjusted for tumor stage (P =.01 and P =.017, respectively). All WTs with high levels of TrkB expression also expressed the brain-derived nerve growth factor ligand. CONCLUSION: Expression of TrkBfull in WT is associated with worse outcome, perhaps because it provides an autocrine survival pathway. Conversely, TrkBtrunc expression is associated with excellent outcome, perhaps as a result of a dominant negative effect.