Detection of N-myc gene expression in neuroblastoma tumors by in situ hybridization.

The presence of N-myc DNA amplification in human neuroblastoma tumors has been shown to be an independent prognostic factor predicting rapid progression of disease. Southern blot analysis has been used previously to detect N-myc amplification in these tumors. The authors report an analysis of N-myc gene expression by in situ hybridization in 28 human neuroblastoma tumors previously studied by Southern blot analysis. In the LA-N-5 human neuroblastoma cell line known to be amplified for N-myc, reaction conditions favoring RNA-RNA hybridization yielded an optimal signal. Using these hybridization conditions, in situ hybridization analysis of N-myc expression in 28 human neuroblastoma tissues correlated perfectly with N-myc DNA amplification in these tumors as detected by Southern blot analysis. In particular, there were no tumors in which N-myc RNA expression was found by in situ hybridization analysis in the absence of DNA amplification detectable by Southern blot, nor were there tumors that had DNA amplification in the absence of RNA expression. Heterogeneity of N-myc RNA expression was observed both among cells in any given tumor area, as well as within different areas of a single tumor. N-myc expression by in situ hybridization analysis was not observed in those tumors with more neuronally differentiated, ganglioneuroma histology. It is concluded that in situ hybridization of tissue sections is as effective as Southern blot analysis of tumor cell DNA in identifying human neuroblastoma tumors in which the N-myc gene is of prognostic significance.     

Modulation of N-myc expression alters the invasiveness of neuroblastoma

N-myc oncogene expression plays a pivotal role in the biology of neuroblastoma, a common childhood tumor. High N-myc expression is associated with advanced disease stage, and in animal models, increased expression results in increased metastatic potential. In normal embryologic development, N-myc expression is associated with neuroblast migration out from the neural crest. To further define the relationship between N-myc and metastasis, an in vitro assay was adapted to measure tumor cell attachment, motility, and proteolytic ability in neuroblastoma cell lines. These parameters were examined in a non-amplified, uniformly N-myc overexpressing cell line and its anti-sense N-myc expressing clones. These lines have been characterized previously, and have a decrease in N-myc expression, growth rate, and tumorigenicity relative to the parent line and vector-only control transfectant. Decrease in N-myc expression resulted in a non-proportional increase of tumor cell attachment, and a proportional decrease in both tumor cell motility and proteolytic ability. In further experiments, assay of a N-myc-amplified overexpressing cell line with an intrinsic heterogeneous pattern of expression demonstrated that motile cells expressed higher amounts of N-myc relative to the general population. Together these relationships indicate that N-myc plays a causative role in the invasive phenotype, and suggest that metastasis may, in part, result from the disruption of a developmentally important normal process.     

Detection of N-myc gene amplification by fluorescence in situ hybridization. Diagnostic utility for neuroblastoma.

We assessed fluorescence in situ hybridization (FISH) as an alternative to Southern blot analysis for determination of N-myc gene amplification in neuroblastoma. In the 44 pediatric solid tumor cell lines examined (20 neuroblastomas), the mean number of N-myc copies determined by FISH correlated closely with Southern blot results. There was wide intercellular variability in gene copy number in tumors that had evidence of amplification; however, tumors judged to be non-amplified completely lacked any cells with high N-myc copy number. FISH provided reliable estimates of N-myc amplification in 12 clinical samples even when the percentage of tumor was low. The other advantages of FISH over Southern blot analysis were speed and technical simplicity, ability to discern heterogeneous gene amplification among tumor cells in the same specimen, and capacity to determine the source of the amplified N-myc signal, whether extrachromosomal double-minute chromosomes, expanded intrachromosomal regions, or chromosome 2 aneuploidy. We conclude that FISH would refine the analysis of N-myc amplification in neuroblastoma and thus improve the assignment of patients to prognostic groups based on this unfavorable risk factor.     

Expression of bcl-2 gene product in neuroblastoma

The expression of bcl-2 is associated with inhibition of apoptosis and prolonged cell survival. The purpose of this study was to examine the immunoreactivity of neuroblastoma and ganglioneuroblastoma tissue samples to the bcl-2 gene product in order to see if it was related to prognosis. BCL-2 protein was detected in all the 46 formalin-fixed, paraffin-embedded samples from 34 patients representing all clinical stages and sites of involvement. Immuno-positivity was observed in tumours from the primary and metastatic sites. Moreover, it was demonstrated in the pre-chemotherapy and the post-chemotherapy samples from six cases with stage 4 disease. It was observed in neuroblasts in various stages of differentiation. A small proportion of undifferentiated neuroblasts were negative. As BCL-2 oncoprotein was present in all the cases irrespective of the clinical outcome, it does not appear to be one of the factors influencing prognosis.     

Increased N-myc mRNA expression associated with dibutyryl cyclic AMP induced neuroblastoma differentiation

The N-myc cellular oncogene is frequently amplified and expressed at a high level in neuroectodermal tumor cells such as neuroblastoma and retinoblastoma. We examined N-myc expression in NCB-20 hybrid (N18TG2 neuroblastoma x embryonic Chinese Hamster brain) cells. After five days of culture, cells treated with 1 mM db cAMP show extensive neurite outgrowth and secrete acetylcholinesterase into the media at a level three times higher than untreated control. In situ hybridizations, dot blots, and Northern analyses reveal four- to eight-fold higher levels of N-myc mRNA in the treated, differentiated cells than in the untreated, undifferentiated controls. Our results show that the highly differentiated state is not incompatible with a high level of N-myc mRNA.     

N-myc amplification and cell proliferation rate in human neuroblastoma

In neuroblastoma, N-myc amplification has been found to be strikingly associated with rapid tumour progression and poor prognosis. Recent studies have demonstrated that cell proliferative activity also significantly predicts the clinical outcome in patients with neuroblastoma. In order to define the correlation between N-myc amplification and cell proliferation rate, in the present investigation the two parameters were first assessed in 48 neuroblastoma tumours. N-myc amplification was evaluated in frozen specimens by Southern-blot analysis using the NB 19-21 probe and it was detected in nine patients. Cell proliferative activity was determined by measuring the AgNOR protein area in histological sections selectively stained by silver. The mean AgNOR protein area value of neuroblastomas with N-myc amplification (3·63±1·62 μm²) was not significantly different from that of neuroblastomas without N-myc amplification (2·46±1·57 μm²; P=0·30). On the other hand, both N-myc amplification and AgNOR protein expression were found to be significantly related to the clinical outcome of the disease (P<0·001 and P=0·0143, respectively; median follow-up time=47 months; range 18–106 months). In a second set of experiments, the relationship between N-myc amplification and cell proliferation rate was assessed in seven established human neuroblastoma cell lines. N-myc amplification was found to be completely independent of the population doubling time (DT), which, on the contrary, was strictly related to the quantitative expression of AgNOR protein (r=−0·947; P<0·001). Altogether, the present results indicate that N-myc amplification and cell proliferation rate are not interrelated in neuroblastoma, each representing an independent biological parameter of cancer cells associated with the clinical behaviour of the disease. © 1997 John Wiley & Sons, Ltd.     

Chromosomal localization of amplified N-myc in neuroblastoma cells using a biotinylated probe.

G-banded chromosome analysis of neuroblastoma cells from two children revealed homogeneously staining regions (hsr) in one patient and double minutes (dmin) in the other. Subsequently, both abnormalities were confirmed as areas of N-myc amplification using chromosomal in situ hybridization with a biotinylated N-myc probe. In addition, the first patient's karyotype contained a possible derivative chromosome 17, which was also demonstrated to contain amplified N-myc, indicating the presence of an hsr unidentified by G-banding. Intercellular heterogeneity in the degree of amplification was also identified in the nuclei of interphase cells. This technique provides a quick method for detecting gene amplification, the identification of which may have useful clinical implications.     

Detection of amplified N-myc gene in neuroblastoma by in situ hybridization using the single-step silver enhancement method

Amplification of the N-myc oncogene in 27 cases of neuroblastoma group tumor was examined by in situ hybridization using the single-step silver enhancement technique. The N-myc gene copy numbers of these neuroblastoma group tumors were previously examined by dot-blot hybridization using DNA extracted from formalin-fixed, paraffin-embedded tissues (H Tsuda et al., Lab Invest, 59:321, 1988). Silver grains were deposited just over the nuclei of tumor cells, but no or only faint deposition was observed over those of infiltrating lymphocytes, stromal fibroblasts, and endothelial cells. We judged a case to be positive for gene amplification when silver grains precipitated over the nuclei of the tumor cells to a greater extent than over nuclei of lymphocytes or endothelial cells in the same section. According to this criterion, 14 cases (12 cases of neuroblastoma and 2 cases of ganglioneuroblastoma) were positive for N-myc gene amplification of 27 cases (18 cases of neuroblastoma, 5 cases of ganglioneuroblastoma, and 4 cases of composite ganglioneuroblastoma). These results corresponded well to those of the dot-blot hybridization analysis using the same materials. Thirteen of 15 cases that carried 4 copies or more of N-myc gene were judged positive, and 11 of 12 cases that carried less than 4 copies of the N-myc gene were judged negative by in situ hybridization. In neuroblastoma group tumors with amplification of the N-myc gene, tumor cells were stained almost homogenously, except for two cases of ganglioneuroblastoma, in which less differentiated small round tumor cells were stained more intensely than differentiated ganglion-like cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

PCNA levels in neuroblastoma are increased in tumors with an amplified N-myc gene and in metastatic stage tumors

N-myc oncogene amplification in neuroblastoma has been found to be significantly associated with advanced stage disease and tumor progression. However, there is a lack of data on tumors, regarding the relationship between N-myc gene amplification and proliferation activity. Proliferating cell nuclear antigen (PCNA) is a proliferation-induced 36 kD nuclear protein that is the auxiliary component of DNA polymerase . PCNA levels in tissues have been found to correlate with proliferative activity. We have examined PCNA levels in neuroblastomas in relation to N-myc gene amplification and tumor stage. Statistically, significantly higher levels of PCNA were observed in tumors with an amplified N-myc gene relative to tumors with a single gene copy. The highest levels of PCNA were observed in advanced stage tumors with an amplified N-myc gene. Treatment of neuroblastoma cells in culture with retinoic acid, which induces differentiation, resulted in a substantial decrease in PCNA. Our results suggest that PCNA levels may reflect differences in proliferative activity between neuroblastomas, related to stage of the disease and to N-myc gene copy number.[/p ]     

Loss of retinoblastoma gene and amplification of N-myc gene in retinoblastoma.

We have analyzed paired samples of genomic DNA from peripheral leukocyte and primary tumor tissue from nine patients with retinoblastoma (RB) and from two RB cell lines, WERI-Rb-1 and Y79, to detect the molecular alterations of the retinoblastoma susceptibility gene (RB-1) and N-myc gene. In Southern analysis, RB-1 deletions in tumor tissues were detected in five patients (56%), one of these revealed a total loss of RB-1. N-myc amplification was found only in one (11.1%) out of nine patients. We also observed a total loss of RB-1 in WERI-Rb-1, and a more than 100-fold amplification of N-myc in Y79. The analysis of the relationship between molecular events and clinical characteristics such as age, sex, tumor laterality did not reveal any specific correlation. These results suggest that genetic backgrounds of RB in Korean patients are quite similar to those of reported cases elsewhere. The high sensitivity of our method in detecting the RB-1 loss indicates that this method can be a useful tool for initially screening a large number of tumors.     

Aberrant expression of p53 gene product in malignant melanoma.

According to the current concept of carcinogenesis, the alterations of p53 tumor suppressor gene have been the most frequently detected in both human cancer cell lines and cancer tissues freshly isolated. This study was conducted to investigate the p53 gene alteration in malignant melanoma. Nineteen tumor tissues were obtained from 19 patients with malignant melanoma and examined for the expression of p53 protein by immunohistochemical staining with mouse monoclonal anti-p53 antibody, NCL-p53-DO-7. Twelve out of 19 cases (63%) showed positive reactions for p53 protein: 26, 21 and 16% of which had low, intermediate and high reactivity, respectively. p53 alteration more frequently expressed in female (10/12) than male patients (2/7) with malignant melanoma (p < 0.05). The incidence of expression of p53 protein was compared according to the stages and the sites of tissue obtained. The positive rate for p53 protein was not significantly different between the stages. The positive rates for p53 protein were five out of five (100%), one out of two (50%) and six out of twelve (50%) in tissues obtained from the metastatic, lymph node, and primary sites, respectively. The difference in the positive rates, however, is not statistically significant. These results suggest that p53 gene is a frequent target for mutation in the development of malignant melanoma.     

Differential expression of delta-like gene and protein in neuroblastoma, ganglioneuroblastoma and ganglioneuroma.

Neuroblastoma is an extremely malignant solid tumor in children, characterized by spontaneous differentiation and regression. An epidermal growth factor-like homeotic protein, delta-like (dlk), has been involved in differentiation of neuroblastoma cell lines, but is unknown in in vivo expression of neuroblastoma. By using in situ hybridization and immunohistochemistry, dlk mRNA and protein expression were studied in formalin-fixed archival tissues from 10 patients with neuroblastoma, five with ganglioneuroblastoma, and five with ganglioneuroma. Three adrenal tissues from children died of diseases other than adrenal tumors and one from an adult with pheochromocytoma were severed as normal and disease controls. The results showed strong immunoreactive dlk staining in endothelial cells in neuroblastoma, ganglioneuroblastoma and ganglioneuroma. Dlk was detectable in mature neuromatous stroma and gangliocytes of ganglioneuroma, but not in neuroblasts of neuroblastoma and ganglioneuroblastoma, neither in gangliocytes of ganglioneuroblastoma. In contrast, dlk mRNA expression was mainly observed in the gangliocytes, but was less intense in the neuroblasts and neuromatous stroma cells. Endothelial cells were essentially devoid of dlk mRNA expression. The findings indicated that there is differential expression of dlk gene and protein among neuroblastoma, ganglioneuroblastoma and ganglioneuroma. The stronger expression of dlk in gangliocytes in ganglioneuroma, in contrast to weaker or no expression in gangliocytes in ganglioneuroblastoma and neuroblasts in neuroblastoma, suggests upregulation of dlk during differentiation of neuroblastoma into more benign form. Furthermore, higher dlk protein expression in the tumor endothelium than in the endothelium of normal adrenal gland implies that dlk may regulate the endothelial function in neuroblastic tumors.     

Analysis of DNA ploidy and proliferative activity in relation to histology and N-myc amplification in neuroblastoma.

Diploid DNA content, advanced stage, unfavorable histology, and N-myc amplification are all associated with aggressive disease and poor prognosis in childhood neuroblastoma. DNA diploidy is associated with advanced stage and unfavorable histology, but the relationships among ploidy, N-myc amplification, and proliferative activity are not known. To determine if DNA diploidy is associated with N-myc amplification, we studied 29 neuroblastomas with flow cytometric analysis and Southern blot analysis. Clinical and histologic features were also evaluated. Sixty percent of the N-myc-amplified tumors were diploid, compared to 26% of the neuroblastomas, which lacked N-myc amplification (P = 0.11). In our analysis of proliferative activity and N-myc amplification, a higher mean percentage of cells in S phase was seen in the N-myc-amplified tumors (13.4%) than in the unamplified tumors (10%), but again the result was not statistically significant (P = 0.14). Significant associations were seen between unfavorable histology and DNA diploidy (P = 0.05), and between unfavorable histology and high proliferative activity (P = 0.007). Our data suggest that biologic factors other than N-myc amplification play a role in determining the aggressiveness of at least some diploid neuroblastomas.     

Hypermethylation-mediated regulation of CD44 gene expression in human neuroblastoma

The CD44 adhesion receptor is silenced in highly malignant neuroblastomas (NBs) with MYCN amplification. Because its functional expression is associated with decreased tumorigenic properties, CD44 behaves as a tumor suppressor gene in NB and other cancers. Given that the precise mechanisms responsible for CD44 silencing are not elucidated, we investigated whether CD44 expression could be regulated by DNA hypermethylation. The methylation status of CD44 gene promoter and exon 1 regions was analyzed in 12 NB cell lines and 21 clinical samples after bisulfite genomic modification, followed by PCR and single-strand conformation polymorphism analysis and genomic sequencing. The results showed that almost all CD44-negative cell lines displayed hypermethylation in both regions, whereas all CD44-expressing cell lines were unmethylated. These observations correlated with the ability to restore CD44 mRNA and protein expression by treatment of CD44-negative cells with the 5-aza-2'-deoxycytidine demethylating agent. In contrast, no CD44 gene hypermethylation could be detected in 21 NB clinical samples of different stages, irrespective of CD44 expression. Although our results suggest that aberrant methylation of promoter and exon 1 regions is involved in CD44 silencing in NB cell lines, they also indicate that methylation of unidentified regulatory sequences or methylation-independent mechanisms also control the expression of CD44 in primary NB tumors and cell lines. We therefore conclude that CD44 silencing is controlled by complex and tumor cell-specific processes, including gene hypermethylation. Further investigation of other mechanisms and genes involved in CD44 regulation will be needed before demethylation-mediated reactivation of the CD44 gene can be considered as therapeutic strategy for neuroblastoma and perhaps other related cancers.     

Human B-lymphocyte precursors do not express the N-myc gene.

N-myc expression has been reported in neuroblastoma, retinoblastoma and small cell lung carcinoma. Increased expression associated with gene amplification in neuroblastoma correlates with disease stage and prognosis. N-myc expression has been observed in diverse murine tissues during early stages of development with loss of expression in later stages. Abelson murine leukemia virus (A-MuLV)-transformed pre-B cells express N-myc, whereas mature B cells do not. To determine whether human B-lymphocyte precursors also have increased N-myc expression, we extracted DNA and RNA from representative cell lines, prepared Southern and Northern blots and examined them with the N-myc probe, pNB-1. RNA from the following B-cell developmental stages were examined. One null, 1 pre-pre-B, 3 pre-B (including pre-B-lymphoblastic leukemia, a poor prognostic category) and 5 mature B. Neuroblastoma cells and tissues served as positive controls; negative controls included human muscle, placenta, epithelial cell lines, monocytic, promyelocytic, and T-cell lines. N-myc expression was detected in neuroblastoma cells, but in none of the mature human B or B-lymphocyte precursor cells. Additional immunocytochemical studies performed for N-myc nuclear protein likewise failed to detect this gene product. We conclude that human pre-B cells, unlike murine B-cell precursors, do not express increased levels of N-myc RNA. Expression of this oncogene in human neoplastic B cells does not appear to correlate with developmental stage or prognostic group.     

神经母细胞瘤MYCN基因扩增和CD44的表达

目的 定量神经母细胞瘤(NB)MYCN基因扩增倍数,分析其扩增情况与NB临床分期、预后的关系;探讨CD44在判断NB预后中的价值。方法 用差示-PCR法(D-PCR)及倍比稀释法检测并定量33例NB标本MYCN基因扩增倍数;用免疫组织化学染色检测NB标本CD44的表达,同时与年龄、临床分期、病理分型、MYCN基因等预后因素作对比分析。结果 (1)10例NB标本存在MYCN基因扩增,临床分期均为Ⅲ期和Ⅳ期,年龄均大于1岁。MYCN基因扩增与患儿的预后差显著相关(P<0.01)。(2)21例CD44呈阳性表达,其中≤1岁、低分期、预后良好组织型、没有MYCN基因扩增的NB患儿CD44阳性率显著增高。CD44阳性病例的2年生存率(57.1％)高于CD44阴性病例(8.3％,P<0.01)。CD44表达级别越高,其2年生存率越高(P<0.01)。结论 (1)MYCN基因扩增与NB 临床高分期及预后差明显相关。(2)CD44阳性表达是判断NB预后良好的较为可靠的一个参考指标;可以作为MYCN基因扩增检测的补充。(3)D-PCR法及倍比稀释法是检测并定量MYCN基因扩增的一种可行的方法,具有简便、标本用量少、灵敏、准确的特点。