Influence of segmental chromosome abnormalities on survival in children over the age of 12 months with unresectable localised peripheral neuroblastic tumours without MYCN amplification.

BACKGROUND: The prognostic impact of segmental chromosome alterations (SCAs) in children older than 1 year, diagnosed with localised unresectable neuroblastoma (NB) without MYCN amplification enrolled in the European Unresectable Neuroblastoma (EUNB) protocol is still to be clarified, while, for other group of patients, the presence of SCAs is associated with poor prognosis. METHODS: To understand the role of SCAs we performed multilocus/pangenomic analysis of 98 tumour samples from patients enrolled in the EUNB protocol. RESULTS: Age at diagnosis was categorised into two groups using 18 months as the age cutoff. Significant difference in the presence of SCAs was seen in tumours of patients between 12 and 18 months and over 18 months of age at diagnosis, respectively (P=0.04). A significant correlation (P=0.03) was observed between number of SCAs per tumour and age. Event-free (EFS) and overall survival (OS) were calculated in both age groups, according to both the presence and number of SCAs. In older patients, a poorer survival was associated with the presence of SCAs (EFS=46% vs 75%, P=0.023; OS=66.8% vs 100%, P=0.003). Moreover, OS of older patients inversely correlated with number of SCAs (P=0.002). Finally, SCAs provided additional prognostic information beyond histoprognosis, as their presence was associated with poorer OS in patients over 18 months with unfavourable International Neuroblastoma Pathology Classification (INPC) histopathology (P=0.018). CONCLUSIONS: The presence of SCAs is a negative prognostic marker that impairs outcome of patients over the age of 18 months with localised unresectable NB without MYCN amplification, especially when more than one SCA is present. Moreover, in older patients with unfavourable INPC tumour histoprognosis, the presence of SCAs significantly affects OS.

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.

Comparative genetic study of intratumoral heterogenous MYCN amplified neuroblastoma versus aggressive genetic profile neuroblastic tumors.

Intratumoral heterogeneous MYCN amplification (hetMNA) is an unusual event in neuroblastoma with unascertained biological and clinical implications. Diagnosis is based on the detection of MYCN amplification surrounded by non-amplified tumor cells by fluorescence in situ hybridization (FISH). To better define the genetic features of hetMNA tumors, we studied the Spanish cohort of neuroblastic tumors by FISH and single nucleotide polymorphism arrays. We compared hetMNA tumors with homogeneous MNA (homMNA) and nonMNA tumors with 11q deletion (nonMNA w11q-). Of 1091 primary tumors, 28 were hetMNA by FISH. Intratumoral heterogeneity of 1p, 2p, 11q and 17q was closely associated with hetMNA tumors when analyzing different pieces for each case. For chromosome 2, 16 cases showed 2p intact, 4 focal gain at 2p24.3 and 8 MNA. The lengths of the smallest regions of overlap (SROs) for 2p gains and 1p deletions were between the SRO lengths observed in homMNA and nonMNA w11q- tumors. Co-occurrence of 11q- and +17q was frequently found with the largest SROs for both aberrations. The evidence for and frequency of different genetic subpopulations representing a hallmark of the hetMNA subgroup of NB indicates, on one hand, the presence of a considerable genetic instability with different SRO of either gains and losses compared with those of the other NB groups and highlights and, on the other hand, the need for multiple sampling from distant and macroscopically and microscopically distinct tumor areas. Narrowing down the different SRO for both deletions and gains in NB groups would be crucial to pinpointing the candidate gene(s) and the critical gene dosage with prognostic and therapeutic significance. This complexity of segmental chromosomal aberration patterns reinforces the necessity for a larger cohort study using FISH and pangenomic techniques to develop a suitable therapeutic strategy for these patients.

Overexpression of the pseudoautosomal gene MIC2 in Ewing's sarcoma and peripheral primitive neuroectodermal tumor.

Ewing's Sarcoma (ES), the second most frequent bone tumor in childhood and adolescence, and the probably closely related peripheral primitive neuroectodermal tumor (pPNET) share a unique cytogenetic translocation between chromosomes 11 and 22. Both of them expose high amounts of a glycoprotein on their cell surface, which can be specifically detected by the mAb HBA-71. The cDNA coding for the HBA-71 antigen was isolated by screening a cDNA expression library constructed from a pPNET-derived cell line. Nucleotide sequencing revealed the HBA-71 antigen to be the product of the pseudoautosomal gene MIC2 previously identified by the mAb 12E7 in haematopoietic cells. This antigen is a glycoprotein with a molecular weight of about 29,000 and is expressed in low amounts in most human cell lines and probably normal tissues and tumors with only a few exceptions. In T-cells the antigen is involved in cell adhesion processes. In ES- and pPNET-derived cell lines MIC2 expression is significantly enhanced. No gross changes in posttranslational modification could be observed. The high expression results in easy and specific detection of the antigen in immunocytochemical analysis of paraffin embedded tissue sections making HBA-71 a useful tool in tumor diagnosis.

International neuroblastoma staging system stage 1 neuroblastoma: a prospective study and literature review.

PURPOSE: To gain insight into the management of non-metastatic neuroblastoma by examining clinical and biologic features of International Neuroblastoma Staging System (INSS) stage 1 tumors. METHODS: Patients were staged by both the INSS and the Evans staging system and were evaluated for biologic prognostic factors. Patients with INSS stage 1 received no cytotoxic therapy. The literature was reviewed for clinical and biologic data about INSS stage 1. RESULTS: We evaluated 10 consecutive patients (median age, 17.5 months) with INSS stage 1; all remain disease-free (median follow-up duration, > 5 years). Tumors were in the abdomen (n = 6), chest (n = 3), or pelvis (n = 1). Neuroblastoma involved margins of resection in six tumors. Poor-prognostic biologic findings included tumor-cell diploidy (n = 2) and unfavorable Shimada histopathology (n = 2). Two patients were to receive chemotherapy for, respectively, a tumor deemed unresectable and a tumor classified as Evans stage III; second opinions resulted in surgical management alone in each case. Published reports confirm that some INSS stage 1 patients (1) are at risk for overtreatment, and (2) have poor-prognostic biologic findings yet do well. CONCLUSION: Surgery alone suffices for INSS stage 1 neuroblastoma, even if biologic prognostic factors are unfavorable, microscopic disease remains after surgery, and tumor size is suggestive of "advanced-stage" status in other staging systems. Attempts to resect regionally confined neuroblastomas should take precedence over immediate use of cytotoxic therapy; otherwise, some patients may receive chemotherapy or radiotherapy unnecessarily.

Survival from locally invasive or widespread neuroblastoma without cytotoxic therapy.

PURPOSE: To test the hypothesis that cytotoxic therapy is not needed at diagnosis to assure the survival of most patients with non-stage 4 neuroblastoma. METHODS: Patients with non-stage 4 disease received no cytotoxic therapy in the absence of N-myc amplification. The International Neuroblastoma Staging System (INSS) was used. RESULTS: Of 84 consecutive patients with previously untreated, newly diagnosed neuroblastoma, 31 (37%) had non-stage 4 disease. All 31 patients initially received no cytotoxic therapy because none of them had N-myc amplification. Nine stage 1 patients are relapse-free. This report focuses on the 22 patients with locally invasive or distant disease: two stage 2A with gross residual tumor postsurgery, 11 stage 2B with ipsilateral or midline lymph node involvement, four stage 3, and five stage 4S. Eight of the 22 patients were older than 1 year. Postsurgery, 13 patients had visible residual disease, and two others had markedly increased urinary catecholamine levels for more than 1 year. Recurrent or enlarging tumors regressed spontaneously (n = 2) or were excised 5 to 39 months after diagnosis (n = 4). One of the latter had chromosome 1p deletions (common in poor-risk neuroblastoma) that were not detected in the patient's original tumor resected 23 months earlier--findings consistent with clonal evolution or multifocal disease. The patient received chemotherapy. All 22 patients are alive 24 to 98 months (median, 64) from diagnosis. CONCLUSION: Our results suggest that non-stage 4 patients without N-myc amplification can be spared cytotoxic therapy because (1) residual postsurgical or recurrent biologically favorable neuroblastoma rarely evolves into lethal stage 4 disease; and (2) neuroblastoma in lymph nodes has no prognostic significance. These findings are remarkable because no other cancer includes subtypes that are curable without therapy to ablate residual disease.

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.

DEC1 expression in 1p-aberrant oligodendroglial neoplasms.

BACKGROUND: Expression of hypoxia-related tissue factors in 1p-aberrant oligodendroglial neoplasms diminishes patient outcome. Differentiated embryo-chondrocyte expressed gene 1 (DEC1) has been described as novel hypoxia-related tissue factor. In our study, we assessed the expression of DEC1 in 1p aberrant oligodendroglial neoplasms and its association with necrosis and expression of hypoxia-inducible factor 1alpha (HIF-1alpha), carbonic anhydrase-9 (CA9), and vascular endothelial growth factor-mRNA (VEGF). MATERIALS AND METHODS: 44 primary and 16 recurrent oligodendroglial neoplasms with 1p-aberrations were investigated immunohistochemically for the expression of DEC1, HIF-1alpha, and CA9. Expression of VEGF was investigated using in situ hybridization. DEC1 expression was correlated with necrosis and with expression of HIF-1alpha, CA9, and VEGF. RESULTS: DEC1 was expressed in tumor cell nuclei, and occasionally in nuclei of endothelial cells, and glial and neuronal cells of surrounding brain tissue. High expression (>10% of tumor cells immunolabeled) of DEC1 was found in 56 cases, low expression (<10% of tumor cells immunolabeled) was found in 3 cases. In 1 case no expression of DEC1 was evident. DEC1 expression showed no topographical association with necrosis or expression of HIF-1alpha, CA9, or VEGF. CONCLUSION: DEC1 expression is found in the majority of 1p-aberrant oligodendroglial neoplasms and does not correlate with necrosis or expression of HIF-1alpha, CA9, VEGF. Thus, immunohistochemical analysis of DEC1 expression is in our hands not suitable for detection of tissue hypoxia in this type of primary brain tumor.

Combined automatic immunological and molecular cytogenetic analysis allows exact identification and quantification of tumor cells in the bone marrow.

PURPOSE: To improve the detection of disseminated tumor cells in bone marrow (BM) and peripheral blood samples of solid tumor patients, a novel computer-assisted scanning system for automatic search, image analysis, and repositioning of these cells was developed. This system allows precise identification and quantification of tumor cells by sequential immunological and molecular cytogenetic analysis. In this study, we attempt to demonstrate the practical use of this approach by analyzing BM samples from neuroblastoma patients. EXPERIMENTAL DESIGN: The disialo-ganglioside (GD2) molecule was used as the immunological target. The GD2 molecule was described as being specific for neuroblastoma cells, although false positive reactions had been suspected. To verify or disprove the neoplastic nature of the immunologically positive cells, sequential fluorescence in situ hybridization was performed on these cells to search for those genetic aberrations found in the corresponding primary tumors. A total of 115 samples from 40 newly diagnosed patients were evaluated for the presence of GD2(+) cells in the BM. RESULTS: GD2 positivity was detected in 95.2% of stage 4 patients, in 100% of stage 4s patients, and in 38.5% of patients with localized/regional disease. In stage 4 and 4s BM samples, the GD2(+) cells were unequivocally identified as tumor cells based on the molecular cytogenetic aberrations found by fluorescence in situ hybridization. However, in BM samples from patients with localized/regional disease, all GD2(+) cells were concluded to represent false positivity due to the absence of genetic aberrations. CONCLUSIONS: Automatic search and sequential molecular cytogenetic analysis of the immunologically positive cells provide precise information on both the number and cytogenetic profile of disseminated tumor cells.

Schwann cells in neuroblastoma.

Why should we consider Schwann cells when we are interested in the biology of neuroblastomas (NBs)? Although we are familiar with the term "stroma-rich" NB, we basically think of a favourable prognostic subgroup, histologically distinguished by the development of a prominent Schwann cell-stroma. According to current opinion on the maturation processes in NBs, the NB-associated Schwann cell is believed to represent a differentiation product of the NB cell, and we therefore do not envisage the Schwann cell as having any important role in NBs. However, our interest was raised after having realised that Schwann cells in NBs are normal cells, very likely attracted to the neoplastic neuroblasts. But what role does this cell play in these tumours? Can we still reduce the appearance of Schwann cells in NBs to an epi-phenomenon or is this cell population responsible for the differentiation of certain NBs? If so, will it be possible to use their strategies to induce differentiation of neuroblasts and so render them non-aggressive, mature ganglionic cells? To shed light on the possible interactions between normal Schwann cells and NB cells, the maturation capacity of NBs and the genetic constitution of the two main cell populations in these tumours are briefly reviewed. Some data leading to the current view on the origin of the Schwann cells in NBs, and several physiological aspects of the Schwann cells, including normal neurone-Schwann cell interactions, are detailed.

Regression and progression in neuroblastoma. Does genetics predict tumour behaviour?

Neuroblastoma (NB) is a heterogeneous disease. The clinical course may range from spontaneous regression and maturation to very aggressive behaviour. Stage 4s is a unique subcategory of NB, generally associated with good prognosis, despite skin and/or liver involvement and the frequent presence of tumour cells in the bone marrow. Another type of NB is the locally invasive tumour without bone and bone marrow involvement which can also have a good prognosis, irrespective of lymph node involvement. Unfortunately, there is only limited biological information on such tumours which have not been treated with cytotoxic therapy despite lymph node involvement, residual tumour mass after surgery and/or bone marrow infiltration. In order to find specific genetic changes common to NBs with a benign clinical course, we studied the genetic abnormalities of these tumours and compared them with highly aggressive tumours. We analysed a series of 54 localised and stage 4s tumours by means of in situ hybridisation performed on fresh cells or on paraffin embedded tissues. In addition, we performed classical cytogenetics, Southern blotting and PCR analysis on fresh tumour tissue. The majority of patients had been treated with surgery alone, and in a number of patients tumour resection was incomplete. Deletions at 1p36 and amplifications of the MYCN oncogene were absent, and diploidy or tetraploidy were not seen in any case, with residual localised tumours possessing a favourable outcome. Unexpectedly, one patient with a tetraploid 4s tumour without any genetic structural changes not receiving any cytotoxic treatment, did well. Interestingly, this genetic spectrum contrasted with that of progressing tumours, in which most had genetic aberrations, the deletion at 1p36 being the most common event. These data, although limited, suggest that an intact 1p36 (recognised by D1Z2), the absence of MYCN amplification and near-triploidy (at least in localised tumours), represent prerequisites for spontaneous regression and/or maturation.

First experience with prognostic factors in unselected neuroblastoma patients. The Austrian Neuroblastoma 87 Study.

Between January 1987 and December 1993, 117 patients were registered in the Austrian A-NB87 study. The male/female ratio was 1.18, with 50 patients below the age of 1 year at diagnosis. Patients were assigned to stage according to the result of primary surgery in localised disease. Age, ferritin and neuron specific enolase were used in addition in stage III disease for risk-adapted treatment. Adrenal or pelvic primary tumour sites were mainly associated (81%) with advanced disease. The median observation time of the study is 3.5 years. The overall survival at 3 years was excellent in low stage disease and IVs patients, i.e. 100% for stage I and IIA (20 patients), 92% in stage IVs (13 patients), 81% in stage IIIA (18 patients) and 69% in stage IIB (8 patients). Stage IV (38 patients) showed a survival rate of 51%, whereas stage IIB (10 patients) had the worst outcome in this study, i.e. 20%, due to treatment-related toxicity. Significant unfavourable prognostic factors were neuron specific enolase (NSE) > 100 ng/ml, ferritin > 300 micrograms/ml and amplified MYCN. This study achieved a better survival rate in stage IV patients and a subgroup of stage III in comparison to our previous study (Pädiatrie und Pädologie 1986, 21, 269) and gives the basis to further reduce treatment intensity in low-risk disease based on biological factors. However, prognosis for high-risk cases was still poor in spite of a very aggressive treatment concept.

Neuroblastoma cells can actively eliminate supernumerary MYCN gene copies by micronucleus formation--sign of tumour cell revertance?

Human neuroblastoma cell lines frequently exhibit MYCN amplification and many are characterised by the presence of morphologically distinct cell types. The neuronal cells (N-cells) and the so-called flat cells (F-cells) are thought to represent manifestations of different neural crest cell lineages and are considered to be the consequence of neuroblastoma cell pluripotency. In this study, various neuroblastoma cell lines were examined for micronuclei. In F-cells of neuroblastoma cell lines with extrachromosomally amplified MYCN, we observed the frequent occurrence of micronuclei. Using fluorescence in situ hybridisation (FISH) with a MYCN specific probe, we demonstrated that these micronuclei were packed with MYCN hybridisation signals. In addition, in a minor percentage of cells, MYCN signals occurred in clusters, adhered to the nuclear membrane and aggregated in nuclear protrusions. In F-cells, a substantial reduction or lack of amplified MYCN copies was observed. These observations let us conclude that extrachromosomally amplified genes can be actively eliminated from the nucleus resulting in a dramatic loss of amplified sequences in the F-cells. Moreover, reduction or loss of amplified sequences in F-cells was shown to be accompanied by downregulation of MYCN expression, by a decrease in proliferative activity and by upregulation of molecules of the major histocompatibility complex class I (MHC I). Interestingly, F-cells are not restricted to neuroblastoma cell cultures, but also occur in cell lines of other tissue origin. All F-cells share important biological features, interpreted as cell revertance, i.e. loss of the malignant phenotype and properties. This fact, together with the demonstration that neuroblastoma cells do not differentiate into Schwann cells in vivo [1] Ambros et al. NEJM 1996, 334, 1505-1511, do not support the hypothesis that F-cells represent Schwannian/glial differentiation in vitro. We therefore postulate that the elimination of amplified MYCN gene copies in cultivated neuroblastoma cells is in line with the phenomenon of tumour cell revertance.

[5 years neuroblastoma screening in Austria: rate of participation, results and a comparison with other screening areas]. / Fünf Jahre Neuroblastom-Screening in Osterreich: Beteiligungsrate, Ergebnisse und ein Vergleich mit anderen Screeningregionen.

In late 1990 a screening program for the early detection of neuroblastoma in infants was introduced in Austria. The program is performed on a voluntary basis in collaboration with general pediatricians and practitioners. Filter strips for urine collection are distributed to parents of infants aged seven to nine months on the occasion of a routine check up. The samples are sent to the laboratory by parents and analysed for vanillylmandelic acid (VMA) and homovanillic acid (HVA). Between January 1991 and December 1995 125,201 infants were screened. The compliance rate was 26.8% for Austria, but great differences were seen for different regions (65% in Carinthia, 10% in Vorarlberg). 30 children were admitted to hospital for investigation of repeatedly elevated urine catecholamines. A neuroblastoma was identified in 16 cases. In 12 of these cases at least one unfavorable prognostic factor was present (stage > or = 3, elevated LDH, unfavorable histology, N-myc amplification, di- or tetraploidy). Neuroblastoma screening of infants aged more than six months seems to detect predominantly those tumors which are unlikely to regress spontaneously. The observation of one false negative case, however, demonstrates that neuroblastomas which become clinically manifest at a later date may remain undetected by early screening. Possible advantages of shifting screening to a later age and repeated screening are discussed.

Induction of senescence in MYCN amplified neuroblastoma cell lines by hydroxyurea.

Recently, it was shown that MYCN amplified cells spontaneously expulse extrachromosomally amplified gene copies by micronuclei formation. Furthermore, it was shown that these cells lose their malignant phenotype and start to age. We tested whether it is possible to encourage neuroblastoma tumor cells to enter the senescence pathway by low concentrations of the micronuclei-inducing drug hydroxyurea (HU). We studied the effect of HU on 12 neuroblastoma cell lines with extra- or intrachromosomally amplified MYCN copies and without amplification. Two extrachromosomally amplified neuroblastoma cell lines (with double minutes) were investigated in detail. Already after 3 weeks of HU treatment, the BrdU uptake dropped to 25% of the starting cells. After 4 weeks, enlarged and flattened cells (F-cells) and increased granularity in the majority of cells were observed. A drastic reduction of the MYCN copy number-down to one copy per cell-associated with CD44 and MHCI upregulation in up to 100% of the HU treated neuroblastoma cells was found after 5-8 weeks. Telomere length was reduced to half the length within 8 weeks of HU treatment, and telomerase activity was not detectable at this time, while being strongly expressed at the beginning. All these features and the expression of senescence-associated-beta-galactosidase (SA-beta-GAL) in up to 100% of the cells support the hypothesis that these cells entered the senescence pathway. Thus, low-dose HU is a potent senescence elicitor for tumor cells with gene amplification, possibly representing an attractive additional strategy for treatment of this subset of tumors.

Pathology and biology guidelines for resectable and unresectable neuroblastic tumors and bone marrow examination guidelines.

The recommendations concerning tumor and bone marrow handling for the evaluation of molecular-biologic and molecular-genetic and immunologic markers presented in this paper were developed by the SIOP Europe Neuroblastoma Pathology and Biology and Bone Marrow Group. Although the Guidelines were developed for neuroblastic tumors (neuroblastoma, ganglioneuroblastoma and ganglioneuroma), they are applicable to all other tumor entities as well. The paper is subdivided in three main parts. The Pathology Guidelines give an overview about the handling, sectioning and securing of tumor material in case of resectable and non-resectable neuroblastic tumors. The Guidelines encompass open biopsies, tru cut biopsies, fine needle aspirations, and bone marrow aspiration. The importance of the pathologic evaluation for the interpretation of the molecular-genetic and molecular-biologic results, which also includes the exact determination of the tumor cell content is stressed. Besides this, recommendations concerning tumor material obtained after cytotoxic therapy, immunohistologic and immuno-cytologic issues and lymph node examination are addressed. In the Biology Guidelines, the different methods for MYCN, chromosome 1p36 investigations and DNA content measurements are discussed and DNA probes are recommended. Furthermore, specified definitions and a common terminology already used in the SIOP Europe Neuroblastoma Group are presented. In the Bone Marrow Guidelines, recommendations concerning the methods to be employed are given and the most important pitfalls are demonstrated. Both the use of standardized methods and the application of a common language will, it is hoped, contribute to the quality and reliability of collected data and thus to a better comparability between and among research reports. These improvements should prove to be of great value for the affected patients.

Detection of Ip36 deletions in paraffin sections of neuroblastoma tissues.

Nonradioactive in situ hybridization (NISH) on sections of paraffin-embedded neuroblastoma tissue was performed to evaluate numerical and structural aberrations of chromosome 1. Two biotinylated probes specific for the heterochromatic (D1Z1) and subtelomeric regions of chromosome 1 (D1S32) were used to study normal tissue and 4 neuroblastoma samples with and without Ip36 deletions. The NISH findings in 3 of the 4 neuroblastomas correlated well with the results obtained by cytogenetic banding analysis. In 1 tumor sample, however, a deletion at Ip36 was observed by NISH, both on metaphase spreads and interphase nuclei, but not by cytogenetics. The NISH method is therefore advantageous when only paraffin-embedded material is available and can be even more sensitive than conventional cytogenetic analyses under certain conditions. Moreover, the technique provides morphological information that cannot be obtained by methods relying on tissue extracts or cell suspensions.

Detection of numerical and structural chromosome abnormalities in pediatric germ cell tumors by means of interphase cytogenetics.

In contrast to the cytogenetically well characterized testicular germ cell tumors (GCT) in adults, reports on cytogenetic studies in pediatric GCT are scarce. The presence of an i(12p) and numerical abnormalities involving chromosome 12 are the most frequent cytogenetic changes in GCT of adults. We have performed in situ hybridization (ISH) studies on paraffin sections and on isolated nuclei of 13 pediatric GCT with particular emphasis on those chromosome abnormalities that are common in adult GCT. These include numerical and structural abnormalities of chromosomes 1 and 12 as well as numerical deviations of chromosomes 8, 10, X, and Y. The histological subsets of the tumors investigated included two dysgerminomas (DGE), one seminoma (SE), two embryonal carcinomas (EC), four mixed and two pure yolk sac tumors (YST), and one undifferentiated (IT) and one differentiated teratoma (TD). Similar to the GCT in adults, additional copies of chromosome 12 were the most frequently observed numerical abnormalities. In contrast to the findings in adult GCT, changes in the size of the pericentromeric hybridization signals of chromosome 12, suggesting the presence of i(12p) chromosomes, were found in only two cases. No chromosome abnormalities were found in the pure TD or in the TD cells of mixed tumors containing a YST component. In the YST portion, however, Ip deletions and/or numerical chromosome changes were present. Surprisingly, deletions of the short arm of chromosome I, del(I)(p36.3), were frequent in pediatric GCT and were the sole abnormality detected in two cases. The Ip36 deletions were present in all stage-IV EC and YST investigated and were absent in the relatively benign TD and in one YST stage-I. Therefore, Ip36 deletions may have value as a prognostic marker in pediatric GCT.

Neuroblastoma cells provoke Schwann cell proliferation in vitro.

BACKGROUND: A subset of human neuroblastomas (NBs) has the capacity to mature completely, imitating sympathetic ganglia. Previously, we showed that the neuronal population in spontaneously maturing NBs usually has a near-triploid DNA content without 1p deletions, and we concluded that the constantly diploid Schwann cells (SCs) do not belong to the neoplastic component of these tumours. We therefore hypothesised that NB cells are able to stimulate SC proliferation, and that SCs trigger NB differentiation. PROCEDURE: We performed in vitro experiments to test this model and to test whether SCs can also influence the growth of aggressive NBs. Human SCs were co-cultivated with NB tumours and cell lines, and were harvested after defined time intervals. Proliferative activity of the SCs and the NB cells was determined by visualisation of 5-bromo-2'-deoxyuridine (BrdU) incorporation or Ki-67 staining. Neurite outgrowth and neurofilament (NF) expression were analysed immunocytochemically and apoptotic rate was determined by a terminal deoxynucleotidyl transferase-mediated dUTP-X fluorescein nick end labelling (TUNEL) assay. RESULTS: Human NB tumours or cell lines unequivocally increased the proliferation of SCs in vitro. In cocultivated NB cells, the proliferative activity was not altered in the first days of cocultivation, although neurite outgrowth and NF expression were enhanced. However, after 10 days, the mitotic rate of neuroblastic cells decreased and the apoptotic rate showed a marked increase. CONCLUSIONS: The results of the cocultivation experiments provide an experimental hint that the in vivo growth of SCs in NBs is caused by the neoplastic neuroblasts, and they also indicate that cells from peripheral nerves can influence the growth of aggressive NB cells if cocultivated.