Search

Background: A comprehensive clinical long-term survey over the complete spectrum of neuroblatoma disease is lacking in the literature., Objective: Our objective was to describe the incidence, risk profiles, therapies, and outcomes for the total cohort of German patients with neuroblastoma including all clinical stages and risk groups., Methods: Epidemiological, clinical, and outcome data of neuroblastoma patients who participated in one of the six consecutive national trials between 1979 and 2015 were analyzed retrospectively., Results: Of all German neuroblastoma patients known to the national childhood cancer registry, ninety seven percent enrolled in one of the trials. The absolute neuroblastoma rate has increased slightly, whereas the median age at diagnosis has decreased. Except for the screening period (1995-2000), the risk factors lactate dehydrogenase (LDH), ferritin, chromosome 1p, and the MYCN oncogene have remained largely constant, with the exception of an increase in MYCN amplification at stage 4 for those aged ≥18 months between trials NB97 (27%) and NB2004 (35%). The 10-year overall survival increased in patients with stage 1-3 neuroblastoma from 83 to 91%, for stage 4S from 80 to 85%, and for stage 4 aged ≥18 months from 2 to 38%. The fraction of patients in stages 1-3 who never received chemotherapy (neither for frontline nor at recurrence) increased from 35 to 60%. The proportion of macroscopically complete surgical resections of the primary tumor decreased for the total population as well as for patients with stage 4 aged ≥18 months. The impact of chemotherapy response on the outcome was trial dependent. The overall proportion of toxic death during the time of the protocol therapy was 6% for stage 4 patients aged ≥18 months and 2% for low-/intermediate-risk patients. The most frequently reported late sequelae in stage 4 patients aged ≥18 months were renal dysfunctions, hypothyroidism, major hearing impairment, and second malignancies., Conclusion: The body of data for incidences, risk profiles, and survival rates from this survey of more than 37 years provides a useful perspective for future studies on neuroblastoma sub-cohorts.

Procedure: To establish the significance of chromosome 17 aberrations in the biology of neuroblastomas, the fresh-frozen material of 53 primary neuroblastomas (average patient age: 20.8 months; stage 1 or 2: n = 10; stage 3: n = 10; stage 4: n = 10; stage 4s: n = 23) was studied by means of comparative genomic hybridization (CGH). Follow-up data were available for 52 of 53 cases studied (average follow-up period: 26.4 months). Except for one, all cases had previously been analyzed for MYCN status (semiquantitative Southern blot analysis). Studies of LOH 1p36 (VNTR-PCR) had been performed on 28 of 53 cases., Results: Chromosome 17 gains were detected in 46 of 53 (86.8%) cases. Whole chromosome gains were mostly restricted to localized tumors (stage 1 or 2: 9 of 10 cases; stage 4s:19 of 23; stage 3: 2 of 10; stage 4:0 of 10 cases), whereas distal 17 gains were significantly associated with clinically advanced tumor stages and patients aged over 1 year at diagnosis. Univariate analyses revealed a statistically significant correlation of distal 17q gains with overall survival (P< 0.01, MYCN amplification: P< 0.01; 1p deletion: P< 0.01) and an elevated recurrency rate (17q: P= 0.02, MYCN amplification: P = 0.05; 1p deletion P= 0.3). There was a strong coincidence of distal 17q gains and 1p deletion or MYCN amplification (P < 0.01)., Conclusion: Our data indicate that distal chromosome 17q gains are of major prognostic relevance for neuroblastoma patients. However, studies on a larger series of tumors have to be performed to assess whether or not these alterations are independent prognostic markers of a poor clinical outcome.

Procedure: Analysis of comparative genomic hybridization (CGH) data of 120 tumors from four different studies, and data of 84 previously unpublishied tumors, allowed delineation of at least six different genetic subsets of neuroblastomas., Results and Conclusions: A small number of tumors show no detectable imballances. A second group of tumors presents with gains and losses of whole chromosomes and is found predominantly in prognostically favorable stage 1 and 2 tumors. The remaining groups are characterized by the presence of partial chromosome imbalances, and are found mostly in stage 3, 4, and 4S tumors. The third group shows 17q gain without 11q loss, 1p loss, or MYCN amplification (MNA). The fourth group has 1p deletion or MNA, and finally, a fifth group shows 11q loss without 1p deletion or MNA, and is found mainly in stage 4 tumors. The latter group is significantly associated with losses of 3p, 4p, and 14q.

The objective of our study was to assess the gene expression of the antiproliferative systems neuronal nitric oxide synthase (nNOS) and adrenomedullin (AM) in human neuroblastoma. A novel real-time PCR method was evaluated using neuropeptide Y (NPY) for validation. Glyceraldehyd-3-phospate dehydrogenase (GAPDH) and NPY gene expression in neuroblastomas of 50 patients were measured in parallel by competitive quantitative and TaqMan real-time RT-PCR. AM and nNOS mRNA were determined by real-time PCR. Our results showed a linear relationship between competitive quantitative and real-time RT-PCR measurements of NPY and GAPDH (r = 0.87 and r = 0.92, respectively). AM and nNOS mRNA was found in all tumor samples. AM/GAPDH mRNA increased with higher differentiation according to Shimada (p = 0.013). There was no relation between MYCN amplification nor with the tumor stage (p = 0.78 and p = 0.30, respectively). AM/GAPDH did not relate to recurrence or death in a 5-year follow-up period. Neuronal NOS/GAPDH expression did not relate to any biological or clinical parameter of prognosis or differentiation. Similar results were obtained when the neuronal marker protein gene product 9.5 (PGP9.5) was used to normalize mRNA concentration. In conclusion, TaqMan real-time PCR appears to be a reliable method to quantify gene expression in neuroblastomas. Adrenomedullin mRNA in neuroblastoma is linked to tumor differentiation but not to prognostic markers., (Copyright 2000 Wiley-Liss, Inc.)

RFLP and microsatellite analysis with 23 polymorphic markers spanning the entire long arm of chromosome 14 in 108 neuroblastomas showed allelic loss in 19 out of 107 (18%) informative tumors, placing 14q among the most frequently affected chromosomal regions in neuroblastoma. One minimal deletion region could be sublocalized in 17 of 19 cases between markers D14S1 and D14S16, and a second one between markers D14S17 and D14S23 in band 14q32. Furthermore, breakpoints in bands 14q23 and 14q12 were detected. These results suggest the presence of at least two putative tumor suppressor gene loci on chromosome 14. Survival analyses revealed no prognostic impact of allelic loss of 14q in neuroblastoma. Genes Chromosomes Cancer 26:40-46, 1999., (Copyright 1999 Wiley-Liss, Inc.)

The objective of this study was to relate plasma neuropeptide Y (NPY) concentrations of patients with neuroblastoma with the stage of the disease, the patients' age, the prognosis, the tumor mRNA expression and with the effect on tumor cell proliferation. Plasma NPY of 85 patients with neuroblastoma was measured by radioimmunoassay. The patients' median age was 18 months (range, three weeks-12 years). Ten children with childhood tumors that did not affect neurological or neuroendocrine structures and ten healthy children served as control groups. NPY mRNA expression in neuroblastoma tissue was assessed by Northern blot analysis. Proliferation of neuroblastoma cells (SK-N-MC and CHP 234) was evaluated by 5-bromo-2'-deoxyuridine incorporation during DNA synthesis in vitro. Plasma NPY levels were significantly higher in stage 3 (p < 0.05), 4 (p < 0.001) and 4S (p < 0.05) patients than in both control groups. Plasma levels above 8 pmol/l were only seen in stages 2 (17%), 3 (32%), 4 (45%) and 4S (44%). Seven of the 12 patients (58%) who died had NPY levels above 8 pmol/l (vs. 29% in survivors; p = 0.05). In patients with longer follow-up monitoring, relapse coincided with increasing NPY levels. There was no relationship between the patients' age and their plasma NPY concentrations (r = 0.08; p = 0.49). No relation was found between NPY mRNA expression in tumor tissue and NPY plasma concentrations of the ten patients (r = 0.08; p = 0.81). NPY in the supernatant of neuroblastoma cells did not alter the 5-bromo-2'-deoxyuridine incorporation during DNA synthesis. In summary, NPY plasma concentrations in patients with neuroblastoma relate to the stage of the disease. The relation to the prognosis is at the threshold of significance. No relation between tissue and plasma NPY, nor any effect of NPY on proliferation of tumor cells was found.

Chromosomal aberrations of 20 stage 4s neuroblastomas were analysed by comparative genomic hybridization (CGH). In a subset of 13/20 tumours, telomerase activity was evaluated by the telomeric repeat amplification protocol (TRAP). The CGH data were compared with the CGH results of ten stage 1 and 2 (stage 1/2) and 22 stage 3 and 4 (stage 3/4) neuroblastomas. A total of 17/20 stage 4s neuroblastomas did not progress clinically, whereas tumour progression with lethal outcome occurred in 3/20 cases. The CGH data of clinically non-progressing stage 4s tumours revealed a high rate of whole-chromosome aberrations (73.4%) with an overrepresentation of mainly chromosomes 2, 6, 7, 12, 13, 17, 18 and an underrepresentation of mainly chromosomes 3, 4, 11, 14. MYCN amplification or 1p deletion was observed in only 1/27 or 2/17 clinically non-progressing stage 4s tumours respectively, whereas all three progressive stage 4s neuroblastomas showed MYCN amplification, 1p deletion and, in 2/3 cases, distal 17q gains. Except for one case, telomerase activity was not observed in non-progressing stage 4s neuroblastomas. In contrast, 4s tumours with lethal outcome revealed elevated telomerase activity levels. Our data suggest that stage 4s neuroblastomas belong to two biologically different groups, one of which displays the genetic features of localized stage 1/2 tumours, whereas the other mimics advanced stage 3/4 neuroblastomas.

The association of molecular characteristics with prognosis has been reported, but not their relationship with each other and their impact in the context of known clinical risk factors. In this study, data of 1249 consecutive intent-to-treat-neuroblastoma patients with more than 1 year follow-up were examined by multivariate analysis using loglinear and Cox proportional hazard regression models on a stage-related basis (stages 1-3: 600, 4S: 116, 4: 533). In a first step, risk factors were identified from 18 selected clinical variables, and risk groups defined. The second step investigated whether molecular characteristics (MYCN, LOH 1p, del 1p, CD44, N-ras, NGF-R, bcl-2, APO-1 (CD95)) contributed additional prognostic information to the model. The loglinear model demonstrated several interactions between clinical factors. By the Cox regression model, seven independent clinical risk factors were found for stages 1-3, seven for stage 4 and two for stage 4S. By subsequent introduction of all molecular variables, MYCN amplification only added significant prognostic information to the clinical factors in localised and stage 4 neuroblastoma. The models allowed the definition of risk groups for stages 1-3 patients by age (e beta = 5.09) and MYCN (e beta = 4.26), for stage 4 by MYCN (e beta = 2.78) and number of symptoms (e beta = 2.44) and for stage 4S by platelet count (e beta = 3.91) and general condition (e beta = 2.99). Molecular factors and in particular MYCN contribute significantly to risk estimation. In conjunction with clinical factors, they are powerful tools to define risk groups in neuroblastoma.

Comparative genomic hybridization (CGH) was applied to 35 neuroblastomas to obtain a global view of genetic imbalances. Results were validated by means of Southern blot hybridization (detection of N-myc amplification), loss of heterozygosity (LOH) studies (detection of deletion 1p), and interphase cytogenetics [dual labelling fluorescence in situ hybridization (FISH) of centromeric 17 and erbB-2]. CGH allowed sensitive detection of N-myc amplification and chromosome 1p deletion, representing the most established prognostic markers of neuroblastoma. In addition, a high rate of chromosome 17 aberrations (63 per cent) with possible prognostic relevance was observed. Previously unreported high level copy number increases indicating oncogene amplification were mapped to chromosome subbands 2p13-14 and 3q24-26. Other recurrent regional chromosomal aberrations were localized on 11q, 12q, 13q, 14q, and 15q. CGH results were fully consistent with data of Southern blot analysis and LOH study, as well as interphase cytogenetics. These results show that CGH is a sensitive method for the detection of all prognostically relevant genetic alterations in neuroblastomas; that CGH considerably simplifies the detection of these alterations, resulting in a single methodological approach; and that CGH is a powerful tool to elucidate previously unknown genetic changes in neuroblastomas.

Tumor specimens of 203 infants with neuroblastomas of different clinical stages-registered in successive multicenter clinical trials of the German Society of Pediatric Oncology-could be examined for N-myc amplification, chromosome 1-ploidy and-structure, CD44 std. expression (in tumor tissue, and also in patient's sera). Eighty-seven (= 43%) of these infants had a non-localized, disseminated neuroblastoma, mainly involving sympathetic nerve tissue, lymph nodes, liver, skin, bone marrow and bones (46 patients were classified into the 4 group, 41 patients in the true 4 group). If the clinical classification between stage 4 and stage 4s was neglected, then 17 of these infants (= 20%) had N-myc amplification (4-64 copies) with 16 already dead. Seven of 9 examined patients with true stage 4- had chromosome 1p aberrations (with N-myc amplification in 5), and among the dead there were 2 with CD44 negative expression. In another series, serum CD44 std. was measured by ELISA, and the highest (significantly different) Kruskal-Wallis mean rank values (147.8) were found in infants (n = 6) with stage 4s compared to the low mean-rank-value of 71.9 in patients with stage 4 (n = 65). Stage 1-3 patients (n = 42) had values of 99.8-88.6. Thus, infants with disseminated neuroblastomas, showing non-diploidy, normal chromosome 1p structure, non-N-myc amplification and high CD44 std. expression in tumor tissue, and also high CD44 std. values in serum, will have the highest chance of survival due to tumor-non-progression. On the other hand, N-myc amplification in the tumor cells was found to be characteristic for stage 4s neuroblastoma patients with tumor progression (n = 6). Therefore, 4s neuroblastoma-patients with N-myc amplified tumors should be aggressively treated like true stage 4 tumor patients.

Neuroblastoma is the most frequent extracranial solid tumor of early childhood. Histologically and genetically, neuroblastoma represents a heterogeneous group of tumors with significant differences in clinical behavior. In the past, several different characteristic chromosomal aberrations of neuroblastoma have been described, of which a deletion on chromosome 1p and N-myc amplification have been shown to be of major prognostic significance. However, the role of various other nonrandom DNA imbalances in tumor development and progression needs to be clarified. Taking advantage of the recently established comparative genomic hybridization (CGH), we show that this method is able to accurately detect chromosomal imbalances of known prognostic impact. As CGH gives a comprehensive picture of genetic imbalances in just one experiment, it additionally sheds light on other abnormalities of possible prognostic relevance. We therefore recommend further use of this method not only in the field of research but also for the purpose of genetic routine diagnostics in neuroblastoma.

Background: Recurrences of neuroblastoma Evans' stage I-III are infrequent events and the types of its progression have rarely been reported. Therefore, we investigated the patterns of progression in a large series of patients with long follow-up., Patients and Methods: The sites of relapse and time to progression and death of 381 consecutive patients in three cooperative trials (NB 79, 82, 85) with follow-up of 5-16 years were analysed. The Southern blot technique was used for N-myc investigation of tumor tissue., Results: Of the 77 relapsing patients, 41 (53%) had local and 36 (47%) systemic recurrences. The relapses occurred in 9 of 76 stage I patients (6 local/3 systemic), in 4 of 82 stage II (1 local/3 systemic) and 64 of 223 stage III neuroblastoma (34 local/30 systemic) patients. The main sites of distant metastasis were bone marrow (41%), lymph nodes (39%) and bone (37%). The median transition time from localised to metastatic neuroblastoma was 13 months and the outcome as poor (overall survival 9 +/- 5%) as that of the primary metastatic disease (14 +/- 3%). Fifty-three children died of tumor progression and 15 patients of treatment-related complications or other non-tumor conditions. The median age at diagnosis was 43 months for the group with systemic relapse compared to 19 months with only local and 10 months without recurrences (p < 0.001). Elevated serum LDH levels at first diagnosis were seen in 81% with metastatic, in 55% with local and in 33% with no tumor progression (p < 0.001). N-myc amplification was found in 4/14 with local and in 6/12 with metastatic recurrences., Conclusion: The high incidence of systemic relapse and the long transition time suggest that transition from localised to metastatic neuroblastoma is not an uncommon pathway.

182 untreated neuroblastomas were examined for the expression of the adhesion molecule CD44s by immunohistochemistry; all tumors were also tested for amplification of the oncogene N-myc by conventional Southern blot analyses. Positive CD44s immunoreactivity correlated not with a more favorable prognosis in contrast to an absence of CD44s expression in stage dependent or independent evaluations. All patients with stage 4S disease (n = 16) expressed CD44s on a high level and had an event-free survival probability of 82%. In undifferentiated neuroblastoma subgroups could be defined, depending on the expression of CD44s (p < 0.01). In 93% of N-myc non-amplified tumors CD44s expression was detected, whereas only 62% of N-myc amplified tumors were positive for CD44s expression. These results point to CD44s expression as a new histological marker in neuroblastoma, having prognostic impact in tumors of undifferentiated morphology.

A comparison of the prognostic impact of five molecular variables in a large series was made, including tests of their nonrandom association and multivariate analysis. Molecular data were available for 377 patients and MYCN amplification, cytogenetic chromosome 1p deletion, loss of chromosome 1p heterozygosity, DNA ploidy and CD44 expression were investigated. Their interdependence and influence on event-free survival was tested uni- and multivariately using Pearson's chi 2-test, Kaplan-Meier estimates, log rank tests and the Cox's regression model. MYCN amplification was present in 18% (58/322) of cases and predicted poorer prognosis in localised (P < 0.001), metastatic (P = 0.002) and even 4S (P = 0.040) disease. CD44 expression was found in 86% (127/148) of cases, and was a marker for favourable outcome in patients with neuroblastoma stages 1-3 (P = 0.003) and 4 (P = 0.017). Chromosome 1p deletion was cytogenetically detected in 51% (28/55), and indicated reduced event-free survival in localised neuroblastoma (P = 0.020). DNA ploidy and loss of heterozygosity on chromosome 1p were of less prognostic value. Most factors of prognostic significance were associated with each other. By multivariate analysis, MYCN was selected as the only relevant factor. Risk estimation of high discriminating power is, therefore, possible for patients with localised and metastatic neuroblastoma using stage and MYCN.

Cell-cell and cell-extracellular matrix interactions mediated by cell adhesion molecules (for example CD44) play an important role in the cascade of metastasis and the progression of human malignant tumours. The most important aim of this review was, on the basis of our results and the literature, to show the correlation between the expression of CD44s and differentiation and prognosis of neuroblastoma. Surprisingly and in contrast to most other malignant tumours, neuroblastomas exhibited an inverse correlation between CD44s expression and tumour progression. It can be stated that CD44s is a prognostic marker in neuroblastoma which correlates significantly with the grade of tumour cell differentiation, but not with clinical stage. Moreover, there exists a statistically significant correlation between MYCN oncogene amplification and the lack of CD44s expression.

48 primary neuroblastomas have been examined for an amplified N-myc and loss of the putative tumor suppressor-gene locus 1p36 by loss of heterozygosity studies (LOH 1p36). In 25% of these tumors N-myc amplification and/or a LOH 1p36 was detected, and these patients had a bad prognostic clinical outcome. 75% of the tumors neither had an amplified N-myc nor a LOH 1p36, and these patients had a much better prognosis. Thus, tumor genetic changes can subdivide two types of neuroblastoma with profound different clinical behavior. Detection of these genetic changes at diagnosis could help in front-line risk adapted therapy stratifications in neuroblastoma.

Neuroblastoma tumors are characterized by aberrations of chromosome 1. Rapid detection of these chromosomal aberrations at diagnosis could give important clues to outcome and therapy. We attempted to detect numerical and structural aberrations of chromosome 1 not only by classical metaphase cytogenetics but also by interphase cytogenetics in order to overcome difficulties of karyotyping due to diminished metaphase quality and quantity in primary neuroblastoma samples. Karyotypic changes of chromosome 1 in 53 primary neuroblastomas were evaluated. In addition, we successfully performed interphase cytogenetics using single and double in situ hybridization procedures with chromosome 1-specific repetitive DNA probes on nuclei preparations obtained from 46 and 20 tumors, respectively. Polysomies of structurally normal chromosomes 1 were predominantly seen in tumors with good prognosis, whereas deletions of 1p material were nearly exclusively confined to progressive tumors. Numerical and structural chromosome 1 aberrations as studied by metaphase and interphase cytogenetics are thus valuable prognostic markers in neuroblastoma.

340 consecutive patients with neuroblastoma stage IV were analyzed for the possible impact of chemotherapy on general condition, remission status, event free survival and survival. The children entered the trials NB 79, NB 82 and NB 85 of the German Pediatric Oncology Society (GPO). The patients did benefit from chemotherapy by considerable improvement of the general condition, by achievement of 30-40% complete and 60-70% partial remissions. The event free survival (EFS) rate 5-8 years after diagnosis was 13% for all 299 protocol patients, the survival (S) rate 10%. The median/mean EFS time were 11.6/23.8 months, the median/mean S time 17.0/29.4 months. The use of response rates as early predictors for long term survival is challenged. Addition of PCVm (cisplatinum, cyclophosphamide, Vm 26) to ACVD (adriamycine, cyclophosphamide, vincristine, dacarbazine) in trial NB 82 resulted in an improvement of the long term EFS rate from 5% to 18% (S rates 7----21%). The introduction of IVp (ifosfamide, VP16) and increase of doses (cisplatinum, Vm 26) did not further improve the results. Maintenance therapy (NB 82) revealed a positive influence on the outcome. Shorter intervals for realization of chemotherapy were associated with a trend for better EFS (NB 85). Although the group of children with bone marrow transplantation showed better EFS and S data compared to the unselected chemotherapy group, the advantage was less clear if matched pairs (remission status at the time of BMT) were compared.(ABSTRACT TRUNCATED AT 250 WORDS)

12 primary neuroblastomas (NB) of different maturation stages, 2 ganglioneuromas (GN), and 2 neuroblastoma cell lines were analysed for RNA expression of the protooncogene N-myc and the gene encoding the nerve growth factor receptor (NGF-r) by Northern-blots, RNA-dot-blots and for receptor presence by immunohistological procedures. In 4 tumors with strongly elevated RNA expression of N-myc the NGF-r RNA expression was weak or absent. In all 8 tumors with highly increased NGF-r transcription no N-myc expression was detectable. These results, indicating an inverse relationship between N-myc and NGF-r expression, could help in establishing markers for differentiation, and thus prognosis, in neuroblastoma.

In 40 children with neuroblastoma of different clinical stages the tumorkaryotype was determined at onset (n = 30) or at relapse (n = 10) of disease after short-term culture of tumor tissues or bone marrow aspirates. None of the 10 stage I, II, and IVs tumors revealed a chromosome 1p aberration, in contrast to stage III and IV tumors where this abnormality was encountered in 25 (=83%) of 30 patients. Amplification of the proto-oncogene N-myc in the tumor-DNA could not be detected in stage I, II and IVs, was however present in 53% of stage III and IV tumors. Cytogenetic phenomena of gene amplification such as Double minutes (DMs) and Homogeneously Staining Regions (HSRs) correlated with N-myc amplification. About 50% of stage III and IV tumors had chromosome numbers in the neardiploid range whereas prognostically favourable tumors were characterized by hyperploid chromosomal numbers mainly in the triploid range. Life-table analysis according to Kaplan-Meier showed a probability of surviving in about 80% of patients with a normal morphology of chromosome 1 in their tumor cells, compared to about 60% in the absence of N-myc oncogene amplification and of about 50%, if aneuploidy is detected. Thus, we think, the presence or absence of chromosome 1p aberration in the tumorkaryotype is the most sensitive discriminator for outcome in children with neuroblastoma.

Chromosome deletion at the short arm of one chromosome 1 (1p32)--the most common aberration in neuroblastoma cells--was found to be combined with the generation of a homogeneously staining region at this specific site in a newly established neuroblastoma cell line (GI-LI-N) from a stage IV neuroblastoma. By in situ hybridization this homogeneously staining region was shown to contain multiple copies of the proto-oncogene N-myc. This 30-fold oncogene amplification was confirmed by Southern-blot and DNA-dot-blot analyses. In two additional cell lines from children with stage IV neuroblastoma (GI-ME-N and GI-CA-N) N-myc amplification was not detected. Chromosome 1, however, was involved in a structural rearrangement in one cell line (GI-ME-N).

An extensive chromosomal analysis was performed in cells of primary tumour and bone marrow metastases from an 8-month-old girl with disseminated neuroblastoma prior to therapy. A modal chromosome number of 46 was found. Consistent abnormalities in the karyotype were seen: in particular, a translocation (1;11), a deletion (2)(p21) and a chromosome 7p+. Furthermore, double minute chromosomes of two distinct sizes were found in 50%-80% of metaphases in the cell populations examined. These extra chromosomal double minutes might be the cytogenetic form of amplified cellular oncogene-DNA.

A new case of primary intracerebral neuroblastoma is reported. The patient, a 2 year old boy, was subjected to temporary successful craniotomy, postoperative irradiation and cytostatic therapy without any influence on the recurrence of the tumor. In contrast to the possible well-known maturation of neuroblastomas a dedifferentiating process occurred. The boy died 8 1/2 months after diagnosis.

When comparing clinical and tumour cytogenetic data on 14 neuroblastoma patients in different stages of disease we found a high incidence of 1p abnormalities (12/12), homogeneously staining regions/double minutes (9/12) and 2p abnormalities (4/12) in 12 unresectable and metastatic tumours (clinical stages III and IV). In contrast, these features were absent in clinical stage II tumours (2/2) with good prognosis. The coincidence of 1p aberrations with poor outcome of disease will be discussed.

Chromosomes were prepared directly from primary tumor and bone marrow aspirates of an 8 months old infant and a 2 years old boy with metastatic neuroblastoma. A total of 765 tumor metaphases were counted after Giemsa-banding and partially karyotyped. "Double minute" chromosomes were found in the metaphases of both primary tumor and metastases of the infant, but only in the primary tumor of the older child. In both patients, structural imbalances (in addition to other translocations) were seen in the short arm of chromosome no. 1. The modal chromosome number was 46 in the infant's primary tumor and bone marrow metastases, but 84 in the primary tumor and 46 and over 100 in the bone marrow metastasis of the older child. Clonal evolution of metastasizing neuroblastoma cells is discussed.

Only 40 out of 104 patients with neuroblastoma survived moronger than 5 years, and 7 longer than 10 years. This indicates a 2 year remission period of 38.4%. Of the 40 surviving children, only 11 (10,4%) showed no further complications, while 29 (72,5%) children suffered from severe later complications originating from the tumor or the therapy. The skeletal system was affected in 42.9%, the peripheral nervous system in 21.5%, and the central nervous system in 19.6%. In 4 patients, we observed a statomotoric retardation and intelligence deficiency, and in 3 other children, an Australian antigenemia. The possible reason of these later complications are discussed in the paper. The high number of such complications in patients with neuroblastoma raises the question whether or not a less radical therapeutic procedure would be more advisable for differentiated tumors.

In 28 patients with neuroblastoma of different stages the karyotype was determined in the primary tumour and/or in the metastases by direct chromosome preparation or short term cell culture. In addition, DNA analysis for the proto-oncogene N-myc was performed for comparison in 10 cases. Abnormalities (deletions, translocations, derivations) of the short arm of chromosome 1 with the most frequent breakpoint at 1p32 (besides rarer aberrations in other chromosomes) were found in the tumour karyotype of 15 of 18 (83%) patients with metastatic disease (stage IV) and in 2 of 3 patients with stage III, but in none of the 7 patients with stages I, II, IV-S who are all alive with no evidence of disease. These 7 surviving patients with good prognosis had a hyperploid tumour karyotype, mainly in the triploid range. Eleven of the 18 (61%) patients with stage IV and 1 of 3 patients with stage III also contained double minutes (DMs) and/or homogeneously staining regions (HSRs) in their tumour karyotypes. N-myc amplification (30 to 60 copies) in the tumour DNA was detected in 2 of 6 (33%) examined cases with stage IV, in 1 out of 2 examined cases with stage III, and correlated with the presence of DMs/HSRs. Life table analysis showed a 90% probability of surviving in patients lacking the 1p abnormality as compared to less than 10% in patients with an aberrant 1p chromosome in the tumour cells. We conclude that tumour karyotype, in particular the structure of the short arm of chromosome 1, is the most important factor in determining the different outcome in children with neuroblastoma.

The history of a 6-year-old girl with a tumor originating from thoracic spine and finally becoming resistant to surgery, radio-, and chemotherapy is reported. Tumor-biopsy material was studied by light and electron microscopy, in cell culture, by acetylcholinesterase ultracytochemistry, and by quantitative catecholamine analysis and this led to the rejection of the initial diagnosis of a neuroblastoma. Light microscopy revealed a uniform population of undifferentiated cells incompletely lobulated by broad fibrovascular septa. Using the electron microscope, cells were characterized by large intracellular pools of glycogen, little cytoplasm with an abundance of free ribosomes and a paucity of organelles. A few cells displayed desmosome-like attachment sites. Staining for specific and unspecific acetylcholinesterase was negative with light and electron microscopy, as were the results of catecholamine histofluorescence using the glyoxylic acid method. The latter result was confirmed by the negative outcome of quantitative analyses of dopamine, noradrenaline, and adrenaline with high pressure liquid chromatography nd electrochemical detection in tissue samples. Tumor cells could easily be maintained in culture for up to 4 weeks. None of a variety of treatments that are known to favor expression of neuronal characteristics in neuroblastoma cells (serum withdrawal, nerve growth factor, dbcAMP, dexamethasone) induced morphological differentiation in cultured tumor cells. On the basis of the clinical history, morphology, and of our experiments with tumor cells, the diagnosis of a so-called extraskeletal Ewing's sarcoma is most likely. Our results strengthen the view that a cell biology approach may be valuable in neuroblastoma differential diagnosis.

From June 1979 to June 1982 72 children with metastatic neuroblastoma older than 1 year of age at diagnosis were treated according to the protocol NB 79 of the German Society for Paediatric Oncology. The chemotherapy included three cycles of adriamycine, cyclophosphamide, vincristine, dacarbazine and five cycles of adriamycine and cyclophosphamide. 34 patients were randomized for additional beta-Interferon (Fiblaferon) treatment (10(5) U/kg daily for 3 weeks followed by 3 times a week for 21 weeks). 38 patients were not treated with Fiblaferon. The response rate was 96% including 35% complete and 61% partial remissions. Preoperative chemotherapy improved remarkably the resectability of the primary tumor without enhancing complication rates. The cumulative proportion surviving was 21% for the interferon group and 6% for the patients not treated with interferon. The cumulative recurrence free survival rate, however, was 5% for both groups. The median survival time was 19 months and the median recurrence free survival time 10 months. A serious side effect of chemotherapy was bone marrow depression (73%) resulting in 1 week delay of drug administration per cycle. The median of administered drug dosages was 87-100% of the recommended dosages. Side effects of beta-Interferon commonly decreased with time and were fever reactions (55%), shaking chills (29%), cardiovascular reactions (16%).(ABSTRACT TRUNCATED AT 250 WORDS)

Chromosomes of two abdominal neuroblastomas (stage II) from a 10-month-old male infant and a 6-year-old girl were analyzed. A modal chromosome number of 70 and 46, respectively, was found. Neither double minute chromosomes (DM) or homogeneously staining regions (HSR), nor visible structural abnormalities of chromosome #1 could be detected. However, a common aberration was found to be a translocation on the short arm of chromosome #19. After tumor resection, both children are alive without any evidence of tumor disease.

Nuclear DNA contents of tumor cells were measures by fluorescence cytophotometry in bone marrow and touch preparations of 26 children with neuroblastoma, registered in the NBL79 study of the German Pediatric Oncology society. In all cases tumor cells were found to have a higher DNA-content as compared to the normal diploid (2c) DNA-value of granulocytes in the same preparation. Average ploidy values of the histograms of bone marrow metastases were in the tetra- to hypertetraploid (4c-5c) range and showed unimodal distribution in the majority of cases. In the primary tumors there was also a wide distribution of DNA-values in the aneuploid range. A bimodal distribution with stemlines at 2,5c and 5c was found in a stage I, grade I, differentiated primary tumor. Various ploidy patterns found at different sites in the large tumor mass of a newborn might be interpreted as an expression of different degrees of differentiation within the same tumor. In 2 of 4 patients where primary tumor and metastatic lesions could be examined there was a higher ploidy in the 5c to 6c, or even in the 8c range in the metastatic cell population. It appears, thus, that clonal evolution of tumor cells occurs during growth and spread of neuroblastoma.

Sixty-four children with neuroblastoma stage I to III c are presented. The coordinated management utilizing surgical excision, irradiation (2--5.000 rad) in stage II and III and multiagent chemotherapy is described. Favorable sites were abdomen (30) and thorax (22). In ten cases the primary site was unknown. The prognosis is influenced by several factors: Patients under 1 year of age without evidence of bone or bone marrow metastases have a favorable outcome (13/15). Nearly all patients with lesions of bone or generalized tumor in bone and/or bone marrow (stage III b and III c) failed to attain long term disease free survival despite combination chemotherapy and the use of radiation therapy (33/34). No relation between histological or biochemical characteristics and prognosis could be found. Biochemical determinations however are useful as an index of response to treatment. Prognosis was independent from sex. A review of the literature and on attempt to improve the therapeutic efficancy in stage III are reported.

Within 1 year 74 children with neuroblastoma were registered, 30 patients with stage I-III (= 41%) and 44 with stage IV-metastatic disease (= 59%). An aggressive chemotherapy regimen employing Adriamycine, Cyclophosphamide, Vincristine, and Dacarbazine yielded 10/24 partial and 9/24 complete remissions after 9 weeks. 5/24 children were treated less than 9 weeks so far. At the end of the chemotherapy protocol (week 33) 6 recurrences were observed; 3 of these children died. 5 patients remained in complete remission, 1 in partial remission. 12/24 of patients were not evaluable because of treatment less than 33 weeks so far. The one year run of the study is too short to evaluate the benefit of Interferon (randomized trial). The toxicity of the regimen is tolerable, including bone marrow depression, vomiting and hyperpyrexia. Breaking off therapy was only necessary in one patient.

A cytogenetic investigation was performed in a family which included 2 individuals with congenital neuroblastomas of the suprarenal gland confirmed by autopsy and one with a ganglioneuroblastoma of the thoracic wall as well as 3 other individuals with tumors which probably were also neuroblastomas. The lymphocytes of the peripheral blood of 5 healthy relatives as well as of the child with the treated ganglioneuroblastoma failed to show a constant alteration of chromosomes. In this family, therefore, the suggestion could not be proofed that the very rare familial aggregation of neuroblastomas is caused by a hereditary chromosomal aberration.

Chromosome analyses including in situ hybridization with the protooncogene N-myc were carried out at diagnosis and several times at relapse in the bone marrow cells of a girl with disseminated neuroblastoma (stage IV) at ages 9, 23, 24, and 26 months, respectively. The tumor karyotype was pseudodiploid exhibiting partial monosomy for the short arm of chromosome #1, an aberration of the short arm of chromosome #2, and double minutes at diagnosis. Further structural aberrations of chromosomes #4, #6, and #17, and homogeneously staining regions could be demonstrated at relapse. In particular, the following structural aberrations were encountered: t(1;?)(p22;?),t(2;?)(p24;?), t(4;6)(q31;q25), and a der(17q). All tumor metaphases without homogeneously staining regions contained double minutes. N-myc, normally positioned within bands 2p23-24, was found to be amplified in a homogeneously staining region on the short arm of chromosome #15 by in situ hybridization. It is speculated that the translocation t(2;?)(p24;?) might be related to N-myc oncogene activation and subsequent amplification.

Fragile sites on lymphocyte chromosomes of 20 patients with neuroblastoma of different clinical stages and histologic differentiation were studied. A hitherto unknown break in the band p13.1 of chromosome #1 was found in a frequency of 3%-15% in nine cases. The same phenomenon was observed in three members of a neuroblastoma family. In another instance, this fragile site was also seen in the mother of a patient. Fragile sites were expressed when cells were cultured in folate-deprived medium and could be slightly enhanced in frequency by aphidicolin. Additional aphidicolin induced possible fragile sites hitherto unknown or not yet accepted by the HGM 8 were detected.

A 4-year-old boy with a stage III abdominal neuroblastoma was studied. Direct chromosome preparation revealed a t(1;?)(p36;?) in three tumor metaphases (one with a chromosome number of 64). After partial resection of the tumor and further tumor shrinkage by intensive combination chemotherapy, the residual tumor mass was removed by a "second-look" operation, and the patient received postoperative radiotherapy to the tumor area. Chromosome analysis from the peripheral blood taken at that time showed tetraploidy in 14% and hypodiploidy in 66% of 50 available metaphases. Structural abnormalities, mainly involving the distal short arm of chromosome 1, could be identified in seven metaphases. A t(1;?)(p36;?) in a diploid blood cell looked identical to the translocation found in the hyperdiploid tumor metaphase. A del(1)(p36) was also found in a blood cell. It is suggested that an association exists between a chromosome fragility at 1p36, in this case postoperatively induced in vivo by chemoradiotherapy, and the development of neuroblastoma.