HETEROGENEITY OF MYCN AMPLIFICATION IN A CHILD WITH STROMA-RICH NEUROBLASTOMA (GANGLIONEUROBLASTOMA)

Amplification of MYCN portends rapid tumor progression and poor prognosis in neuroblastoma. MYCN copy number has been described as homogeneous within a tumor and congruent in primary tumor and metastasis. We report a child with stage III favorable histology stroma-rich neuroblastoma (ganglioneuroblastoma) and a poor outcome with an apparent change in MYCN gene amplification by Southern blot. Initial biopsy revealed a ganglioneuroblastoma with predominance of differentiating cells designated as neuroblastoma, stroma-rich, intermixed (Shimada). Southern blot failed to demonstrate MYCN gene amplification. After front-line chemotherapy failed, a total resection was performed. In this specimen, Southern blot demonstrated MYCN amplification (15-20 copies) in the undifferentiated component and no amplification in the differentiated. Fluorescence in situ hybridization (FISH) analysis performed retrospectively on both tumor biopsies demonstrated MYCN amplification in the undifferentiated sections of both tumor specimens but not in the differentiated ones. This is the first well-documented case report of heterogeneous MYCN amplification in a child with neuroblastoma. Because key therapeutic decisions are based on the presence of MYCN amplification, physicians diagnosing and treating children with neuroblastoma need to be aware of the possibility that MYCN amplification may be heterogeneous within a tumor and may be missed using techniques based on pooled DNA such as Southern blotting. FISH may be a preferable method for determining MYCN amplification.     

17q gain in neuroblastoma predicts adverse clinical outcome. U.K. Cancer Cytogenetics Group and the U.K. Children's Cancer Study Group

BACKGROUND: It is now recognized that gain of chromosome 17 material is the most frequent genetic abnormality of neuroblastoma cells. Several studies have linked 17q gain with known adverse prognostic factors: patient age >1 year, advanced stage disease, deletion of chromosome arm 1 p, and amplification of the MYCN oncogene. We sought to further investigate the clinical and prognostic associations of chromosome 17 status in relation to other well-established predictive factors. PROCEDURE: In a collaborative study by UK cytogenetics centres, we compiled a series of 104 neuroblastoma tumours for which the status of chromosome 17 was confidently defined by cytogenetics, metaphase or interphase FISH, or CGH analysis. The results were correlated with data on 1p and MYCN, and with centrally collated clinical and survival information. RESULTS: Gain of 17q (i.e., unbalanced gain of segment 17q21-qter) was found in 66.3% of tumours, while 33.7% showed a '17q normal' status (i.e., no gain at all, or gain of whole chromosome 17 relative to ploidy). Gain of 17q was strongly associated with advanced stage disease, patient age >1 year, 1p deletion, and MYCN amplification (all P< 0.01). In univariate analysis, 17q gain was a significant predictor of adverse outcome (projected 5 year relapse-free survival 15.6% compared to 75.2% in cases lacking this feature in tumour cells; (P < 0.0001). In multivariate analysis, 17q gain was more strongly associated with adverse outcome than was either stage (Stage 4 vs other combined) or 1p status. CONCLUSION: We conclude that gain of chromosome segment 17q21-qter is of great biological and clinical importance in neuroblastoma, and that its detection at diagnosis should be a priority.     

Genetic analysis of p73 localized at chromosome 1p36.3 in primary neuroblastomas

BACKGROUND: Human p73, a novel homolog of p53, has recently been cloned and mapped at chromosome 1p36.3, the locus for putative tumor suppressor gene(s) of neuroblastoma (NBL) and other cancers. p73, like p53, inhibits growth and induces apoptosis in neuroblastoma and osteosarcoma cell lines. PROCEDURE: To test the hypothesis that p73 is a NBL suppressor gene, we examined expression, allelo-typing, and mutation of the p73 gene in primary human neuroblastomas. Loss of heterozygosity (LOH) for p73 was performed in 272 primary NBLs using a CT repeat polymorphic marker, which we found in intron 9 of the p73 gene. RESULTS: p73 LOH was observed in 28 out of 151 (19%) informative cases. The high frequency of p73 LOH was significantly associated with sporadic neuroblastomas (P< 0.001), MYCN amplification (P< 0.001), and advanced stages (P< 0.05). Mutational analyses by PCR-SSCP (single strand conformation polymorphism) revealed two mis-sense mutations in 140 NBLs, one somatic and one germline. CONCLUSION: Thus, the present results have shown that mutation of p73 is infrequent in NBLs, although the p73 locus is frequently lost in advanced stage tumors. These suggest that p73 may not be a tumor suppressor in the classic Knudson manner.     

Increased expression of the MYCN oncogene in human neuroblastoma cells and possible, new therapeutic approaches

Human neuroblastomas of advanced stages often display amplification with a consecutive enhanced expression of the MYCN oncogene. Enhanced MYCN expression is thought to contribute in a causative manner to the progression of neuroblastomas, but the mechanisms by which this may occur have remained unclear. By transfecting human neuroblastoma cells that display a normal MYCN expression with the human MYCN oncogene, we have generated a cell line with enhanced MYCN expression and thereby were able to compare the biological and biochemical properties of the transfected and non-transfected cells. We have demonstrated autocrine growth factors in the MYCN-transfected, but not the non-transfected, neuroblastoma cells. Identification of the primary structures of these factors may help to develop specific antagonists in order to improve the therapy of advanced neuroblastomas. Currently, this could be done by application of genistein or tumor necrosis factor. As we could demonstrate for the first time, the dietary constituent genistein is able to inhibit the proliferation of neuroblastoma cells with enhanced and normal MYCN expression, but also that of cells derived from other solid pediatric tumors. In contrast, tumor necrosis factor is able to inhibit selectively the proliferation of neuroblastoma cells with enhanced MYCN expression. We suggest that tumor necrosis factor might improve the therapy of advanced human neuroblastomas.     

Concomitant p53 mutation and MYCN amplification in neuroblastoma

The MYCN oncogene is amplified in 20% of childhood neuroblastoma and is associated independently with poor prognosis. Alteration of the p53 tumor supressor gene, in contrast, occurs infrequently in these tumors. In this report, we described a 3-year-old girl with stage IV neuroblastoma. Molecular analysis revealed both MYCN gene amplification and a point mutation of the p53 tumor supressor gene. To our knowledge, this is the first reported case of neuroblastoma with genetic alterations of both these genes. Med. Pediatr. Oncol. 29:206–207, 1997. © 1997 Wiley-Liss, Inc.     

荧光定量RT-PCR 检测神经母细胞瘤细胞MYCN基因mRNA的表达

目的：MYCN基因表达对神经母细胞瘤的治疗及预后评估有指导意义,目前国内对于MYCN基因mRNA的定量检测未见报道,该研究拟采用SYBR 绿色荧光染料Ⅰ(SYBR GREEN Ⅰ) 实时检测的逆转录-聚合酶链反应（RT-PCR）方法,检测神经母细胞瘤细胞系LA-N-5细胞 MYCN基因mRNA的表达,并对其可行性及实用性进行研究,力争探索出微量瘤标本的MYCN基因mRNA定量检测的可行方法。方法：提取神经母细胞瘤细胞系LA-N-5 细胞总RNA,采用SYBR GREEN I 实时检测的RT-PCR检测其MYCN基因mRNA的表达,并用3-磷酸甘油醛脱氢酶(GAPDH)作为内参照,将MYCN基因的mRNA拷贝数与GAPDH的拷贝数相除,结果为单细胞MYCN基因mRNA的表达水平。结果：反应标准曲线有良好的相关性( R2>0.99), PCR 产物特异, 神经母细胞瘤细胞系LA-N-5 MYCN基因mRNA的表达水平为17.4±1.2。结论：只要严格控制PCR 反应条件,SYBR GREEN Ⅰ定量RT-PCR 法可以作为一种良好的定量PCR方法对神经母细胞瘤细胞系LA-N-5 MYCN基因mRNA的表达进行检测,此方法为临床微量神经母细胞瘤瘤组织的MYCN基因mRNA的定量检测提供了可能。［中国当代儿科杂志,2007,9（1）：47-50］     

苦参碱抑制神经母细胞瘤LA-N-5细胞增殖及MYCN基因mRNA的表达

目的：神经母细胞瘤是4岁以下儿童最常见的恶性实体肿瘤,MYCN基因的高表达导致预后更加恶劣。苦参碱作为中药苦参的重要成分对多种肿瘤有治疗作用,该实验拟应用苦参碱作用神经母细胞瘤细胞(LA-N-5),对肿瘤细胞增殖及MYCN基因mRNA表达受抑制情况进行初步研究,希望可以为神经母细胞瘤的治疗开拓新的思路。方法：以终浓度为0.25 mg/mL,0.50 mg/mL,0.75 mg/mL,1.00 mg/mL苦参碱作用神经母细胞瘤LA-N-5细胞, MTT法检测LA-N-5细胞增殖情况变化,采用SYBR 绿色荧光染料Ⅰ的实时荧光定量RT-PCR检测MYCN基因mRNA表达受抑制情况。结果：苦参碱对LA-N-5细胞增殖的抑制呈明显的剂量、时间依赖性,随剂量的增加,作用时间的延长,抑制效果逐渐加强。1.00 mg/mL作用72 h后肿瘤细胞增殖抑制效率达36.3%,单细胞MYCN基因mRNA表达9.7±0.35拷贝,抑制效率达44.6%。结论：应用苦参碱在体外环境作用LA-N-5细胞,可以有效抑制该细胞的增殖、原癌基因MYCN mRNA的表达,为临床上神经母细胞瘤的治疗开拓了新的思路。     

Survivin mRNA levels are associated with biology of disease and patient survival in neuroblastoma: a report from the children's oncology group

Alterations in apoptotic mechanisms favoring cell survival may be vital for modifying tumor behavior. Survivin, an inhibitor of apoptosis, and caspase 8, a proapoptotic enzyme, are key players in cellular apoptotic mechanisms. We investigated whether the levels of survivin and caspase 8 and the ratio between these 2 apoptotic factors correlate with tumor biology and predicts outcome in patients with neuroblastoma. Survivin and caspase 8 levels were quantified in 38 primary tumor specimens and analyzed individually and in relation to each other. High survivin expression and high survivin:caspase 8 ratios were associated with MYCN amplification, unfavorable histology, and high-risk group of disease (P<0.0008). High survivin mRNA levels were associated with worse overall survival (P=0.02) although the median follow up was only 22.6 months with a range of 1 day to 3.3 years. Low caspase 8 expression was associated with stage 4 disease, high-risk group, MYCN amplification, and unfavorable histology. Although the survivin:caspase 8 ratio was associated with these risk factors, the ratio did not improve the predictive value of survivin alone in this small series. Quantifying multiple apoptotic genes in neuroblastoma may supplement current risk stratification. Moreover, categorizing aberrant apoptotic gene expression in neuroblastoma may translate into novel therapeutic targets.     

Rapid and accurate determination of MYCN copy number and 1p deletion in neuroblastoma by quantitative PCR

MYCN amplification and 1p36 deletion are adverse prognostic factors in neuroblastoma, and rapid accurate determination of MYCN amplification is essential for risk stratification. MYCN copy number and 1p36 deletion status were determined by fluorescence in situ hybridization (FISH) and real time PCR in a diagnostic pathology laboratory setting on 35 consecutive patients with neuroblastoma. The PCR technique was technically successful in all cases and results were generally available within 24 hr of biopsy. There was no discordance between FISH and PCR results. Real time PCR is a reliable, accurate, and simple technique that can be applied to small neuroblastoma biopsies allowing rapid diagnosis.     

RNA干扰抑制神经母细胞瘤细胞MYCN基因的初步研究

目的对RNA干扰抑制神经母细胞瘤细胞（LAN-5）MYCN基因表达进行初步研究。方法体外化学合成针对MYCN基因的小干扰RNA（siRNA），经脂质体Lipofectamine 2000包裹后转染神经母细胞瘤细胞，以无关siRNA和未转染的细胞为对照，采用SYBR绿色荧光染料Ⅰ的实时荧光定量RT-PCR检测siRNA的抑制效果。结果脂质体转染siRNA效率可达84.83％，化学合成的siRNA可抑制神经母细胞瘤细胞MYCN基因mRNA表达，转染72h后抑制率达58．3％。结论化学合成的siRNA可以有效抑制神经母细胞瘤MYCN基因的表达，为神经母细胞瘤基因治疗开辟了新的途径。     

Intensified chemotherapy increases the survival rates in patients with stage 4 neuroblastoma with MYCN amplification

PURPOSE: Patients with high-risk neuroblastoma who have multiple copies of MYCN fare much worse than do those without MYCN amplification; however, it has not been clarified whether intensified chemotherapy with or without blood stem cell transplantation can alter the extremely poor prognosis of patients with amplified MYCN. METHODS AND RESULTS: Between 1985 and 1999, 301 patients older than age 12 months with stage 4 neuroblastoma were treated. From January 1985 to February 1991, 80 patients with stage 4 neuroblastoma with and without MYCN amplification uniformly received induction chemotherapy with regimen A(1) (cyclophosphamide 1,200 mg/m(2) and vincristine 1.5 mg/m(2) on day 1, tetra-hydropyranyl [THP]-Adriamycin 40 mg/m(2) on day 3, and cisplatin 90 mg/m(2) on day 5). Among 22 patients with MYCN amplification, nine (40.9%) achieved a complete remission and seven (31.8%) underwent stem cell transplantation. Of 58 patients without MYCN amplification, 43 (74.1%) achieved a complete remission and 14 (24.1%) underwent stem cell transplantation. The 5-year relapse-free survival rates were 23.2% for stage 4 patients with MYCN amplification and 33.3% for those without MYCN amplification (P = 0.029); the 5-year overall survival rates were 32.8% for stage 4 patients with MYCN amplification and 42.8% for those without MYCN amplification (P > 0.05). From March 1991 to June 1998, patients with stage 4 neuroblastoma who had 10 or more copies of MYCN were treated with regimen A(3) (cyclophosphamide 1,200 mg/m(2) per day on days 1 and 2, THP-Adriamycin 40 mg/m(2) on day 3, etoposide 100 mg/m(2) per day on days 1 to 5, and cisplatin 25 mg/m(2) per day on days 1 to 5); those with fewer than 10 copies of MYCN received regimen new A (cyclophosphamide 1,200 mg/m on day 1, THP-Adriamycin 40 mg/m on day 3, etoposide 100 mg/m per day on days 1 to 5, and cisplatin 90 mg/m on day 5), which is similar in intensity to regimen A. Among 88 patients with MYCN amplification, 63 (71.6%) achieved a complete remission and 63 (71.68%) underwent stem cell transplantation. Of 133 patients without MYCN amplification, 93 (69.9%) achieved a complete remission and 71 (53.4%) underwent stem cell transplantation. The 5-year relapse-free survival rates were 36.0% for stage 4 patients with MYCN amplification and 32.2% for those without MYCN amplification (P > 0.05), the 5-year overall survival rates were 34.0% for stage 4 patients with MYCN amplification and 38.9% for those without MYCN amplification (P > 0.05). The difference in relapse-free survival rates was significantly different (P = 0.003) between patients with MYCN-amplified tumor treated before (regimen A(1)) versus after 1991 (regimen A(3)). CONCLUSIONS: With the use of the more intensive induction regimen A plus blood stem cell transplantation for MYCN-amplified patients, survival curves for those with or without MYCN amplification now appear similar. Higher doses of chemotherapy may ameliorate the effect of MYCN amplification in patients with high-risk neuroblastoma.     

Neuroblastoma in southern Brazil: an 11-year study

The authors report on the incidence and clinical characteristics of neuroblastoma in southern Brazil. The aims of the study were to evaluate the age at diagnosis, tumor stage, MYCN status, and tumor histopathology, and to relate these factors to survival. All patients with neuroblastoma, 15 years old or younger (n = 125), admitted to the three major pediatric oncology hospitals in the state of Parana over a period of 11 years (between January 1990 and December 2000), were included in the analysis. All patients were followed for at least 5 years. In addition, a FISH evaluation for MYCN status was conducted in a subset of 34 tumors. Overall survival for tumor stages 1, 2, 3, and 4 was 100%, 72%, 59%, and 17%, respectively. Sixty-two percent (77/125) of all patients were older than 2 years; these represented 71% (57/80) of the patients with stage 4 disease. Children who presented with an unfavorable histopathology had a significantly worse prognosis (20% survival) than children with a favorable histopathology (67% survival). MYCN amplification was detected most commonly in stages 3 and 4 tumors (13/16). These data showed a delayed diagnosis of neuroblastoma in children in southern Brazil, and consequently survival was considerably lower in these patients.     

Genomic profile in high risk neuroblastoma by comparative genomic hybridization

Different subtypes of neuroblastoma (NB) carry associated genetic aberrations that predict their clinical course. Whole chromosome gains are usually associated with early clinical stages and good prognosis, while 1p deletion, 17q gain and MYCN amplification (MNA) are related to advanced stages and poor prognosis. High-risk neuroblastomas (NB-HR) include NB in children aged more than 1 year old, either stage 4 or any stages showing MNA except stage 1. The prognosis of NB-HR patients remains poor, despite aggressive therapy. Only MNA confers poor prognosis. Between January 2000 and February 2005, tumoral specimens from 60 patients with NB-HR were sent to the Spanish Reference Center for NB biological studies. In all cases, MYCN together with 1p36 status was analyzed by fluorescence in situ hybridization (FISH). Comparative genomic hybridization (CGH) was performed in 24 cases. Using FISH we detected 31 MNA cases including 29 with 1p36 deletion; there were 21 cases without MYCN amplification (MNNA) but 7 of these had 1p36 deletion; 8 cases showed MYCN gain (MNG) but 6 of these had 1p36 deletion. CGH showed other chromosomal alterations. Of 11 MNA cases, none had 11q loss and all of them showed 17q gain or 17 disomy. Of the 7 MNNA cases, there were 4 with 11q loss including 2 with 3p loss and all presented 17q gain or 17 disomy. The 6 MNG cases included 4 cases with 11q loss and 5 cases with 17q gain or 17 disomy. Genomic profiling by CGH in NB-HR confirms the interaction among genetic alterations, the prognostic significance of which should be evaluated to establish new treatment criteria.     

Biological characteristics of neuroblastoma with partial deletion in the short arm of chromosome 1

BACKGROUND: Neuroblastoma shows remarkable heterogeneity, resulting in favorable and unfavorable outcomes. It is well known that almost all cases with MYCN amplification have a poor prognosis. We have previously reported that unfavorable tumors show high telomerase activity, whereas favorable tumors show low or nil activity. We also found that the unfavorable neuroblastoma often have a loss of heterozygosity (LOH) at the MYCL locus. PROCEDURE: To clarify the biological and clinical profiles of tumors with genetic abnormalities of the short arm of chromosome 1, we performed deletion mapping on 1p on 92 neuroblastoma tissues and corresponding noncancerous samples obtained from 92 cases for 24 micro- or minisatellite loci. RESULTS: LOH was detected in at least one locus of 1p in 43 (47%) cases. All samples were classified into four groups according to the deleted pattern: interstitial deletion (group I, n = 20), short terminal deletion (group ST, n = 6), large terminal deletion (group LT, n = 17), and without detectable deletion (group N, n = 49). All group I cases, whose SRO (shortest region of overlap) was at 1p36.1-2, survived disease free, and none of them showed MYCN amplification or high telomerase activity except for one case. On the other hand, in group LT cases, who showed a large terminal deletion from D1S162 (1p32-pter), including the SRO of group 1, only 5 out of 17 have survived disease free, and 13 showed MYCN amplification or high telomerase activity. The six group ST cases showed small terminal deletion from 1p36.3 with modest prognosis, similar to the group N. CONCLUSIONS: Thus, we propose three loci, 1p36.1-2, 1p32-34, and 1p36.3, as the candidate loci of neuroblastoma suppressor genes on chromosome 1p responsible for groups I, LT, and ST, respectively. Among them, the 1p32-34 locus may be associated with aggressiveness of tumor progression, possibly due to MYCN amplification and/or telomerase reactivation, while the remaining two loci may not.     

Detection of MYCN DNA in the cerebrospinal fluid for diagnosing isolated central nervous system relapse in neuroblastoma

We present the case of a 1-year-old female with stage-4 neuroblastoma with MYCN amplification; she was treated with five chemotherapy courses, resulting in normalization of elevated serum levels of tumor markers. Complete remission was achieved after allogeneic hematopoietic stem-cell transplantation with reduced-intensity conditioning. Nine months later, however, the tumor relapsed in the central nervous system (CNS). The serum and cerebrospinal fluid (CSF) levels of the tumor markers were normal, but the MYCN copy number was high only in the CSF DNA, suggesting an isolated CNS recurrence. The MYCN copy number in the CSF DNA was reflective of response to treatment.     

Association between congenital cardiovascular malformations and neuroblastoma

OBJECTIVE: We explored the association between neuroblastoma and congenital cardiovascular malformations (CCM), previously described in case reports. STUDY DESIGN: Echocardiogram and chart reviews of a series of 158 patients with neuroblastoma and a control group of 192 children with leukemia were performed. The proportion of patients with CCM in each group was compared. RESULTS: Fourteen of the 70 (20%) patients with neuroblastoma and echocardiography had CCM, compared with 7 of the 192 (3.6%) patients with leukemia with echocardiograms (P=.0001). If all of the patients with neuroblastoma without echocardiograms (n=88) are considered to have normal cardiac anatomy, this difference remains significant (14 of 158 patients with neuroblastoma have CCM detected [8.9%] versus 7 of 192 patients with leukemia [3.6%]; P=.045). Neural crest-derived CCM were more common in patients with neuroblastoma, detected in 5 of 70 patients with neuroblastoma versus 2 of 192 patients with leukemia (P=.016). Congenital cardiovascular malformations in patients with neuroblastoma were associated with a cancer diagnosis at age less than 1 year and a lower neuroblastoma stage, but there was no association with tumor MYCN amplification. CONCLUSIONS: Neuroblastoma and CCM may be associated. We recommend echocardiography for CCM screening in patients with newly diagnosed neuroblastoma.     

Prognostic value of MYCN and ID2 overexpression in neuroblastoma

BACKGROUND: The molecular mechanisms driven by overexpression of the MYCN gene in neuroblastoma are not well known. Whether MYCN overexpression in the absence of genomic amplification, or ID2 overexpression has prognostic value remains controversial. PROCEDURE: Ninety-nine neuroblastic tumors from Memorial Sloan-Kettering Cancer Center and 12 neuroblastoma cell lines were analyzed by Affymetrix U95v2 Microarray System. The expression levels of the genes MYCN and ID2 were determined by RT-PCR and immunohistochemistry and the clinical value of the overexpression of both genes was determined. RESULTS: MYCN genomic amplification with overexpression was prognostic for survival (P = 0.0005). Stage 4 patients with MYCN amplification but without overexpression, had no increased likelihood of death, whereas cases with MYCN overexpression but no genomic amplification showed a low survival (P = 0.0096). ID2 did not correlate with MYCN expression (R = -0.23 for tumors and -0.27 for cell lines) or with survival (P = 0.8746). CONCLUSIONS: MYCN amplification was not related to clinical outcome in the absence of overexpression in neuroblastoma tumors. ID2 expression appears to be independent of MYCN expression and lacks prognostic value.