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.

FKHRL1-mediated expression of Noxa and Bim induces apoptosis via the mitochondria in neuroblastoma cells.

Protein kinase-B (PKB) and its target, the forkhead transcription factor like 1 (FKHRL1)/FoxO3a, have been suggested as regulators of neurotrophin-mediated cell survival in neuronal cells. We analyzed human neuroblastoma cells and found that FKHRL1 was phosphorylated, suggesting its inactivation. To study FKHRL1 function, we infected SH-EP and NB15 cells with a 4OH-tamoxifen-regulated FKHRL1(A3)ER(tm) transgene. Activation of FKHRL1 promoted cytochrome-c release and caspase-dependent apoptosis. FKHRL1 induced TRAIL and the BH3-only proteins Noxa and Bim, implicating both extrinsic and intrinsic death pathways. However, expression of dnFADD did not inhibit FKHRL1-induced cell death, whereas Bcl2 protected against apoptosis. This excluded the death-receptor pathway and suggested that cell death decision is regulated by Bcl2-rheostat. Importantly, RNAi knockdown of Noxa or Bim decreased apoptosis, indicating that Noxa and Bim cooperate to mediate FKHRL1-induced cell death. We conclude that Noxa and Bim establish a connection between FKHRL1 and mitochondria, and that both BH3-only proteins are critically involved in FKHRL1-induced apoptosis in neuroblastoma.

Treatment of localised resectable neuroblastoma. Results of the LNESG1 study by the SIOP Europe Neuroblastoma Group.

Main objective of this study was to confirm that surgery alone is an effective and safe treatment for localised resectable neuroblastoma except stage 2 with amplified MYCN gene (MYCNA). Of 427 eligible stages 1-2 patients, 411 had normal MYCN and 16 had MYCNA. Of the 288 stage 1 patients with normal MYCN, 1 died of complications and 16 relapsed, 2 of whom died; 5-year relapse-free survival (RFS) and overall survival (OS) rates were 94.3% (95% confidence interval (CI): 91.6-97) and 98.9% (95% CI: 97.7-100), respectively. Of the 123 stage 2 patients with normal MYCN, 1 died of sepsis and 22 relapsed, 8 of whom died (RFS 82.8%, 95% CI: 76.2-89.5; OS 93.2%, 95% CI: 88.7-97.8). In stage 2, OS and RFS were worse for patients with elevated LDH and unfavourable histopathology. Of 16 children with MYCNA, 7 were stage 1 (5 relapses and 4 deaths) and 9 were stage 2 (3 relapses and 2 deaths) patients. In conclusion, surgery alone yielded excellent OS for both stage 1 and 2 neuroblastoma without MYCNA, although stage 2 patients with unfavourable histopathology and elevated LDH suffered a high number of relapses. Both stage 1 and 2 patients with MYCNA were at greater risk of relapse.

Localization of Agrobacterium rhizogenes T-DNA in plant chromosomes by in situ hybridization.

We have used in situ hybridization to determine the sites of insertion of Agrobacterium rhizogenes Ri T-DNA in the chromosomes of Crepis capillaris (2n = 6) transformed roots. Four transformed root lines were obtained by infecting Crepis stem segments with A. rhizogenes. Southern hybridization analysis indicated that each root line was the result of one or more independent T-DNA insertion events. In two root lines, one copy of T-DNA was present; the other two root lines each contained two copies of T-DNA. To localize these T-DNA inserts on Crepis chromosomes, metaphase spreads were perpared from each root line, and hybridized in situ to a biotinlabeled T-DNA probe. The results indicated that T-DNA was present in a different chromosomal location in each root line, and that each chromosome had been a target for T-DNA insertion at least once. In the root lines containing two T-DNA inserts, two patterns of integration were observed: in one case the T-DNAs were present on separate chromosomes; in the other case the two T-DNAs were close together (but not tandemly arranged) on a single chromosome. A comparison of these results and those obtained previously for a fifth Crepis-transformed root line demostrated that Ri T-DNA does not insert preferentially into a particlar chromosomal location.

Sequence based high resolution chromosomal CGH.

We aimed to directly align a chromosomal CGH (cCGH) pattern with the gene mapping data by taking advantage of the clustering of the GGCC motif at certain positions in the human genome. The alignment of chromosomal with sequence data was achieved by superimposition of (i) the fluorescence intensity of the sequence specific fluorochrome, Chromomycin A3 (CMA3), (ii) the cCGH fluorescence intensity profile of individual chromosomes and (iii) the GGCC density profile extracted from the Ensembl genome sequence database. The superimposition of these three pieces of information allowed us to precisely localize regions of amplification in the neuroblastoma cell line STA-NB-15. Two prominent cCGH peaks were noted, one at 2p24.3, the position 15.4 mega base (Mb), and the other at 2p23.2, 29.51 Mb. FISH and high resolution array CGH (aCGH) experiments disclosed an amplification of MYCN (16 Mb) and ALK (29.2-29.9 Mb), thus confirming the cCGH data. The combined visualization of sequence information and cCGH data drastically improves the resolution of the method to less than 2 Mb.

Adenovirus E1A does not induce the Ewing tumor-associated gene fusion EWS-FLI1.

Rearrangement of the EWS gene with FLI1 is thought to occur early in the pathogenesis of Ewing's sarcoma family tumors (EFTs) because the chromosomal aberration is pathognomonic for this disease. Recently, adenovirus (Ad) 5 E1A protein has been reported to induce this gene rearrangement in a variety of cell types. This finding, if generally substantiated, not only suggests an etiological role for viral agents in the generation of oncogenic chromosomal aberrations but would also significantly impact the use of adenoviral vectors for gene therapy. In contrast, we now report on the absence of EWS-FLI1 chimeric products from short- and long-term cultures of stably Ad-transformed cells lines and from transiently E1A-expressing cell lines. In addition, we demonstrate the absence of E1A from EFTs. We conclude that there is no role for Ads in EFT pathogenesis. Consequently, evidence for a viral genesis of tumor-specific gene rearrangements is not available.

The EWS protein is dispensable for Ewing tumor growth.

EWS encodes a ubiquitously expressed RNA binding protein with largely unknown function. In Ewing sarcoma family tumors (EFT), one allele is rearranged with an ETS gene. This is the first description of an EFT with a complete EWS deficiency in the presence of two copies of a rearranged chromosome 22 carrying an interstitial EWS-FLI1 translocation. Absence of EWS protein suggested that it is dispensable for EFT growth. By sequencing of EWS cDNA from unrelated EFTs, we excluded inactivation of EWS as a general mechanism in EFT pathogenesis. Rather, EWS was found to be uniformly expressed in two splicing variants of similar abundancy, EWSalpha and EWSbeta, which differ in a single amino acid. Three EWS negative cell lines were established, which will serve as valuable models to study normal and aberrant EWS function upon reintroduction into the tumor cells.

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.

A novel putative tyrosine kinase receptor with oncogenic potential.

We have detected transforming activity by a tumorigenicity assay using NIH3T3 cells transfected with DNA from a chronic myeloproliferative disorder patient. Here, we report the cDNA cloning of the corresponding oncogene, designated UFO, in allusion to the as yet unidentified function of its protein. Nucleotide sequence analysis of a 3116bp cDNA clone revealed a 2682-bp-long open reading frame capable of directing the synthesis of a 894 amino acid polypeptide. The predicted UFO protein exhibits characteristic features of a transmembrane receptor with associated tyrosine kinase activity. The UFO proto-oncogene maps to human chromosome 19q13.1 and is transcribed into two 5.0 kb and 3.2 kb mRNAs in human bone marrow and human tumor cell lines. The UFO locus is evolutionarily conserved between vertebrate species. A 4.0 kb mRNA of the murine UFO homolog is expressed in a variety of different mouse tissues. We thus have identified a novel element of the complex signaling network involved in the control of cell proliferation and differentiation.

Co-amplification of a novel gene, NAG, with the N-myc gene in neuroblastoma.

Substantial evidence implicates amplification of the N-myc gene with aggressive tumor growth and poor outcome in neuroblastoma. However some evidence suggests that this gene alone is not the sole determinant of outcome in N-myc amplified tumors. We have searched for genes that co-amplify with N-myc in neuroblastoma by means of two-dimensional analysis of genomic restriction digests. Using this approach, we have identified and cloned a novel genomic fragment which is co-amplified with N-myc in neuroblastomas. This fragment was mapped in close vicinity to N-myc on chromosome arm 2p24. It was amplified in 5/8 N-myc amplified neuroblastoma cell lines and in 9/13 N-myc amplified tumors. Using a PCR-based approach we isolated a 4.5 kb c-DNA sequence that is partly contained in the genomic fragment. The open reading frame of the cDNA encodes a predicted protein of 1353 amino acids (aa). The homology of the predicted protein, which we designated NAG (neuroblastoma amplified gene), to a C. elegans protein of as yet unknown function, and its ubiquitous expression suggest that NAG may serve an essential function. By Northern blot analysis we showed that amplification of the cloned gene correlates with over-expression in neuroblastoma cell lines. Amplification and consequent over-expression of NAG may, therefore, contribute to the phenotype of a subset of neuroblastomas.

Mutational hot spots within the carboxy terminal region of the LMP1 oncogene of Epstein-Barr virus are frequent in lymphoproliferative disorders.

We have recently identified in Epstein-Barr virus (EBV) positive Hodgkin's disease (HD) a variant of the latent membrane protein 1 (LMP1) oncogene characterized by four point mutations and a 30 base pair deletion. These findings led us to test whether such mutants were also present in other lymphoproliferative disorders (LPD). We analysed 98 EBV DNA positive cases (67 LPD, 15 benign conditions, 16 lymphoblastoid cell lines) by PCR for deletions within the LMP1 gene. DNA sequencing of the region coding for the carboxy terminal protein domain was performed on 24 cases. In 13 cases the same combination of 4 point mutations at positions 168,320, 168,308, 168,295 and 168,225 was identified. Of these cases, 12 had an additional point mutation at position 168,357 and eight at position 168,355, and nine had a 30 base pair deletion including nucleotides 168,285 to 168,256. These deletion mutants were identified in HD, angioimmunoblastic lymphadenopathy, B-immunoblastic lymphoma, peripheral T-cell lymphoma, and two lymphoblastoid cell lines. Our findings reveal a high frequency of non-random point mutations at preferential sites within the 3' (carboxy terminal) region of the LMP1 oncogene. The association of these mutational hot spots with LPD suggests that they are involved in EBV related lymphomagenesis and that they define a clinically relevant EBV strain.

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.

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.