The role of MYCN and PHOX2B gene combined with plasma cell-free DNA detection in risk stratification and predicting the prognosis of high-risk neuroblastoma / 中华实用儿科临床杂志

Objective:To explore the clinical significance of the MYCN gene, PHOX2B gene and plasma cell-free DNA (cfDNA) in risk stratification and predicting the prognosis of high-risk neuroblastoma (NB). Methods:This was a prospective study involving 94 high-risk NB children admitted to Beijing Children′s Hospital, Capital Medical University from August 2017 to December 2018.Relative levels of MYCN and PHOX2B and cfDNA at diagnosis, and 4 and 6 cycles of chemotherapy were detected, and their differences were compared by the Chi- square test.Kaplan-Meier survival analysis was performed to explore their prognostic potential in high-risk NB. Results:Among the 94 high-risk NB children, 14 cases (14.9%) had MYCN amplification, 76 cases (80.8%) had positive expression of PHOX2B and 56 cases (59.6%) had cfDNA level higher than 100 μg/L.The proportion of high lactate dehydrogenase (LDH, ≥1 500 U/L) level in the MYCN gene amplification group (6/14 cases) was higher than that in the normal group (9/80 cases) ( P=0.009). The proportion of multi-site metastasis (54/76 cases) and high neuron specific enolase (NSE) level (NSE≥370 μg/L, 37/76 cases) in PHOX2B positive group were significantly higher than those in the negative group (5/14 cases, 2/14 cases) ( P=0.015, 0.020). The proportion of high LDH and high NSE in high cfDNA concentration (≥229.6 μg/L)group (13/37 cases, 28/37 cases) were significantly higher than those in low cfDNA concentration group (2/48 cases, 10/48 cases) (all P<0.001). With the decreased tumor burden during the treatment, the copy number of PHOX2B gene and cfDNA level were significantly lower than those at the initial diagnosis [0 (0-719.6) copies vs.1 723.5 (0-186 000.0) copies; 19.0 (1.1-225.5) μg/L vs.200.6 (8.0-5 247.4) μg/L, all P<0.001]. The 2-year event-free survival (EFS) rate of the MYCN gene amplification group was significantly lower than that of the normal group[(33.3±13.1)% vs.(58.5±7.1)%, P=0.020]. The 2-year EFS rate of PHOX2B positive group was significantly lower than that of the negative group[(47.9±7.1)% vs.(79.1±11.1)%, P=0.043]. EFS rate in high cfDNA concentration group was significantly lower than that in cfDNA low concentration group[(38.6±9.8)% vs.( 71.7±8.2)%, P=0.001]. After 6 cycles of chemotherapy, EFS rate in the PHOX2B positive group was significantly lower than that in the negative group [(16.7±14.4)% vs.( 60.6±6.6)%, P=0.014]; which was significantly lower in the Metaiodobenzylguanidine (MIBG) positive group than that of the negative group[(35.2±11.7)% vs.(65.8±7.1)%, P=0.037]. The MYCN gene and cfDNA concentration were not correlated with the prognosis of high-risk NB.Survival analysis of the combination of PHOX2B and MYCN gene ( PHOX2B+ /MIBG + , PHOX2B+ or MIBG + , PHOX2B-/MIBG -) showed a significant difference in the survival among three groups[0 vs.(53.6±1.2)% vs.(65.5±7.4)%, P=0.003]. Conclusions:The MYCN and PHOX2B gene and cfDNA concentration are of significance in risk stratification and predicting the prognosis of high-risk NB.Compared with the MYCN gene and cfDNA concentration, the PHOX2B gene is more suitable for monitoring the curative effect of chemotherapy on high-risk NB.A combined analysis of PHOX2B gene and MIBG before treatment can be more accurate in evaluating the treatment effect and residual lesions.

Effects of miR-144 on proliferation, apoptosis and cisplatin resistance by targeting MYCN in pediatric neuroblastoma / 中华肿瘤杂志

Objective@#To investigate the effects and mechanisms of miR-144 on proliferation, apoptosis and cisplatin (DDP) resistance of neuroblastoma cells.@*Methods@#Real-time fluorescence quantitative PCR (RT-qPCR) was used to detect the mRNA expressions of miR-144 and MYCN in neuroblastoma cell lines, including SH-SY5Y and SK-N-SH, and human umbilical vein endothelial cells HUVEC. The miR-negative control, miR-144 mimics, si-negative control, si-MYCN, miR-144 mimics and pcDNA, miR-144 mimics and pcDNA-MYCN co-transfected SH-SY5Y cells were described as miR-NC, miR-144, si-NC, si-MYCN, miR-144+ pcDNA and miR-144+ pcDNA-MYCN group, respectively. The half maximal inhibitory concentration (IC50) and cell proliferation were detected by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H tetrazolium bromide (MTT) assay. The protein expressions of MYCN, p21, cyclin D1, Bax, Bcl-2 were analyzed by western blot. Cell apoptosis was detected by flow cytometry. The cell fluorescence activity was detected by double luciferase reporter gene assay.@*Results@#Compared with HUVEC cells, the expressions of miR-144 in neuroblastoma cells SH-SY5Y and SK-N-SH significantly decreased, while the mRNA and protein expression of MYCN significantly increased. The IC50 of DDP was 9.16 μg/ml in SH-SY5Y cells. The absorbance value in 490nm (A490 value) of miR-144 group was 0.30±0.03, significantly lower than 0.46±0.03 of miR-NC group. The cell apoptotic rate of miR-144 group was 26.94%±2.01%, significantly higher than 9.68%±0.52% of miR-NC group. The IC50 value of DDP in miR-144 group was 2.95±0.26, significantly lower than 9.23±0.61 of miR-NC group. The expressions of p21, cyclin D1, Bax, Bcl-2 in miR-NC and miR-144 group were 2.67±0.19, 0.41±0.04, 2.12±0.21, 0.18±0.01 and 1.01±0.07, 1.00±0.06, 1.00±0.05, 1.00±0.06, respectively, with statistical significance (all P<0.05). Knockdown of MYCN showed the similar effects with those of miR-144 overexpression in SH-SYSY cells. MiR-144 significantly inhibited the fluorescence activity of ectopic MYCN expressing cells and negatively regulated the expression of MYCN. Overexpression of MYCN can reverse the effects of miR-144 on proliferation inhibition, apoptosis promotion and sensitization of SH-SY5Y cells to DDP.@*Conclusion@#MiR-144 inhibits proliferation, promotes apoptosis and enhances the sensitivity of neuroblastoma cells to DDP through targeting MYCN, which provides a potential treatment for neuroblastoma.

Differential Clinical Significance of Neurotrophin-3 Expression according to MYCN Amplification and TrkC Expression in Neuroblastoma

BACKGROUND: Neurotrophin-3 (NT-3), a member of the NT family, has only been considered an ancillary compound that provides anti-apoptotic benefits by inactivating tropomyosin receptor kinase C (TrkC)-induced apoptotic signals. However, little is known about the clinical relevance of NT-3 expression itself in neuroblastoma. The purpose of this study was to assess NT-3 expression in patients with neuroblastoma and its relevance to clinicopathologic findings and treatment outcomes. METHODS: In this study, expression of NT-3 and TrkC was analyzed using immunohistochemistry in 240 patients with newly diagnosed neuroblastoma. RESULTS: The results of the study revealed that NT-3 expression was associated with older age at diagnosis, localized tumors, and more differentiated tumors but was not associated with early treatment response (degree of residual tumor volume after three cycles of chemotherapy) and progression-free survival (PFS). However, when analysis was confined to patients with MYCN amplified tumors, NT-3 expression was associated with better early treatment response with borderline significance (P = 0.092) and higher PFS (86.9% vs. 58.2%; P = 0.044). In multivariate analysis in patients with MYCN amplified tumors, NT-3 was independent prognostic factor (hazard ratio, 0.246; 95% confidence interval, 0.061–0.997; P = 0.050). In another subgroup analysis, the early treatment response was better if NT-3 was expressed in patients without TrkC expression (P = 0.053) while it was poorer in patients with TrkC expression (P = 0.023). CONCLUSION: This study suggests that NT-3 expression in neuroblastoma has its own clinical significance independent of TrkC expression, and its prognostic significance differs depending on the status of MYCN amplification and/or TrkC expression.

Synergistic anti-tumor effect of metformin and dichloroacetate in MFCW-ampIified neuroblastoma BE-2C cells / 中国药理学通报

To investigate the effect of metformin production of combined group compared with alone combined with dichloroacetate on the proliferation of A/FC/V-amplified neuroblastoma BE-2C cells and its mechanisms. Methods The inhibitory effects of metformin and dichloroacetate alone or in combination on BE-2C cells was measured by CCK-8 assay; the concentrations of glucose and lactic acid in the medium were measured by a glucose test kit and L-lactic acid assay kit; cell apoptosis was determined by flow cytometry (FCM) with Annexin V-FITC conjugated propidium iodide (PI) staining; the expressions of apoptosis related protein were detected by Western blot. Results Metformin and dichloroacetate showed significant proliferation inhibition activity on BE-2C cells; there was obviously decreased glucose uptake and lactic acid production of combined group compared with alone group (P <0. 01) ; the apoptotic rate was significantly higher in combined group compared with that in alone group (P <0. 01) ; Bax and cleaved caspasce-3 protein expression markedly increased in combined group, but Bcl-2 protein expression significantly increased compared with alone group (P <0. 01). Conclusions Metformin combined with dichloroacetate shows a significant synergistic anti-tumor effect against BE-2C cells by reducing the accumulation of lactic acid caused by metformin and inducing apoptosis.

Gene research progress of retinoblastoma / 中华实验眼科杂志

Retinoblastoma (RB) is a common intraocular malignant tumor of infants.It not only seriously threats children's eyesight,but also endangers their lives.RB develops from the immature cells of retina and its occurrence is closely related with the tumor suppressor gene RB1.The inactivation of two alleles of RB1 is the basis of RB occurrence and development.With the rapid development of biological technology,RB gene related research has made great progress.Researches showed that there are many changes in the chromosome level of RB.Many genes are also involved in the development and progression of RB,including oncogene MYCN,murine double minute 4 (MDM4,also known as MDMX),driver protein family members 14 (K IF14),DEK,E2F3,tumor suppressor gene calcium 11 (CDH11) and so on.This review summarizes the progress in gene research of RB,reveal pathogenesis of RB on DNA molecular level and provides a scientific basis for clinical doctors to formulate effective therapeutic plans.

Detection of MYCN Amplification in Serum DNA Using Conventional Polymerase Chain Reaction

Neuroblastoma (NB) is the most common extra-cranial solid tumor of childhood and is characterized by a wide range of clinical behaviors. Amplification of MYCN is a well-known poor prognostic factor in NB patients. As the MYCN amplification status is usually tested using tumor specimens, lengthy and invasive procedures are unavoidable. To evaluate the possibility of detecting MYCN amplification without invasive procedure, we performed conventional polymerase chain reaction (PCR) analysis to identify MYCN amplification using the preserved serum DNA. PCR of serum DNA was done in 105 NB patients whose MYCN status had been confirmed by fluorescence in situ hybridization. MYCN amplification was evaluated as the ratio of signal intensities between MYCN and NAGK (M/N ratio). When regarding the tissue FISH results as a reference, 10 patients had MYCN-amplified (MNA) NB, and 95 had non-MNA NB. The M/N ratio of the MNA group (median 2.56, range 1.01-3.58) was significantly higher than that of the non-MNA group (median 0.97, range 0.67-5.18) (P < 0.001). In the receiver operating characteristic curve analysis, the area under the curve was 0.957 (95% confidence interval 0.898-1.000; P < 0.001), and it showed 90.9% sensitivity and 97.9% specificity with the selected cut-off value set as 1.6. The detection of MYCN amplification using conventional PCR analysis of serum samples seems to be a simple and promising method to evaluate the MYCN status of NB patients. Further study with a larger set of patients is needed to confirm the accuracy of this result.

Study on 13-cis retinoic acid inducing differentiation of three types of human neuroblastoma cell lines in vitro / 中华实用儿科临床杂志

Objective To investigate the effects of 13-cis retinoic acid (13-cis RA) in inducing differentiation of 3 types of human neuroblastoma (NB) cells in vitro.Methods The status of MYCN gene amplification of cultured SH-SY5Y,SK-N-SH and SK-N-BE2 cells was detected by fluorescence in situ hybridization.After treatment with different concentrations of 13-cis RA,morphological changes were observed by phase-contrast microscope,and neuron-specific enolase (NSE) concentrations were determined by enzyme linked immunosorbent assay.The cell viability was measured through cell counting kit-8 assay,and the cell apoptosis was assayed with flow cytometry (FCM).Results The morphological changes in differentiation were observed in all 3 types of NB cells after 13-cis RA treatment.MYCN amplification was detected in SK-N-BE2 cells even after 13-cis RA treatment,while the other 2 cell lines were amplification-null.After different concentrations of 13-cis RA treatment,NSE concentration increased with prolonged time,especially for SK-N-BE2 cell(F =27.00,P ＜ 0.000 1).13-cis RA stimulated cell proliferation within 48 hours of treatment,and then inhibited cell growth.FCM indicated that the degree of apoptosis in SH-SY5Y cell was significantly higher after 13-cis RA treatment of 10 μmol/L concentration for continuous 96 h and 120 h as compared to the control group (F =16.21,P =0.011;F =16.04,P =0.016).Cell apoptosis of SK-N-SH cell after 13-cis RA treatment of 1 μ mol/L and 10 μ mol/L concentration for 48 h,were significantly higher than those of the control group (F =15.05,P =0.012;F =31.18,P =0.005);while SK-N-BE2 cell with different concentrations of 13-cis RA(1 μmol/L,5 μmol/L,10 μ mol/L) for 120 h were significantly higher than those of the control group(F =9.05,P =0.030;F =11.38,P =0.028;F =7.88,P =0.041).Conclusions The present study showed that 13-cis RA could induce differentiation of human NB ceils in vitro.It induces cell proliferation within 48 hours of 13-cis RA,and thereafter suppresses cell growth.No improvement was found in MYCN amplification cells with the detection of DNA level after 13-cis RA treatment,which suggests that combined treatment is possibly needed.

The unduced effect of arsenic trioxide in different types of neuroblastoma cell lines / 中华实用儿科临床杂志

Objective To identify the effect of arsenic trioxide (As2O3) on the differentiation and apoptosis of different types of neuroblastoma(NB) cell lines.Methods The cell lines [SK-N-SH,SK-N-BE2,SH-SY5 Y] were induced with different concentrations (0 μmol/L,1 μmol/L,3 μmol/L,5 μ mol/L and 7 μ mol/L) of arsenic trioxide for 24 h,48 h,72 h under the same conditions.The expression of MYCN gene was examined by fluorescence in situ hybridization assay in SK-N-BE2,cell proliferation,cell cycle and cell apoptosis were detected with cell counting kit-8 (CCK-8) assay and flow cytometry.Results 5 μmol/L of As2O3 inhibited the expression of MYCN gene in SK-N-BE2;CCK-8 assay indicated that As2O3 inhibited the proliferation of NB cell in a dose-and time-dependent manner,the cell proliferation was significantly suppressed compared with the low concentration (1 μ mol/L) after treated with As2O3 by 1 μmol/L,3 μmol/L,5 μmol/L and 7 μmol/L in 24 h,48 h and 72 h,SH-SY5Y:24 h(chisq =9.666 7,P ＜ 0.05),48 h (chisq =9.666 7,P ＜ O.05),72 h (chisq =9.512 8,P ＜ 0.05);SK-N-SH,24 h (chisq =10.38,P＜0.054 6),48 h(chisq=8.641 0,P＜0.05),72 h(chisq=9.461 5,P＜0.05);SK-N-BE2:24 h (chisq =8.435 9,P ＜0.05),48 h(chisq =8.641 O,P ＜0.05),72 h(chisq =9.545 5,P ＜0.05);compared with the control group,the As2O3-treated cells showed increased apoptosis percentage,with the percentage of 1.6％ (0 μmol/L),3.8％ (1 μmol/L),6.1％ (3 μmol/L),10.4％ (5 μmol/L),40.2％ (7 μ mol/L);the cell cycle was arrested at G2/M phase,which prevented cell division.Conclusions (1) As2O3 play an important role on the NB cells proliferation,apoptosis which were dose-and time-dependent manner.(2)As2O3 can inhibit the expression of MYCN gene.(3)As2O3 also could block NB cell cycle at S and G2/M,and inhibit the cell nucleus replication and the As2O3 had different induced effect between different types of NB cell.

Detection of MYCN gene amplification in neuroblastic tumors by fluorescence in situ hybridization and its clinical significance / 中华实用儿科临床杂志

Objective To explore the significance of MYCN gene amplification in children with neuroblastic tumors(NT).Methods The clinicopathological data of 154 cases with NT were reviewed,including general data, classification of pathology,clinical stage and prognosis.MYCN gene amplification was detected by fluorescence in situ hybridization(FISH) and its relationship between pathological characteristics and prognostic significance was analyzed.Results There was 154 cases of NT aged 1 day to 11 years,with a mean age of 26.1 months,and the median age of 20.5 months.Male and female ratio was 1.48 : 1.00.According to International Neuroblastoma Staging System (INSS) ,20 cases were of stage Ⅰ (13.0％) ,23 cases of stage Ⅱ (14.9％) ,43 cases of stage Ⅲ (27.9％) ,64 cases of stage Ⅳ(41.6％) and 4 cases of Ⅳs (2.6％).There were 72 cases(46.8％) with favorable histology,and 82 cases(53.2％) with unfavorable histology.MYCN amplification was found in 20 cases (13.0％) and the signal ratio of MYCN and chromosome 2 (CEP2) was 4.08-43.29.One hundred and thirty-four cases of MYCN non-amplification included MYCN gain in 91 cases(68.0％) ,MYCN negative in 43 cases(32.0％).MYCN expression showed the significant differences in ages, neuroblastoma type, international neuroblastoma pathology classification (INPC), mitosis karyorrhexis index (MKI), and clinical stages (all P ＜ 0.05).No significant difference was found in gender(P ＞ 0.05).Of 20 MYCN amplification cases,4 cases (20.0％) survived and 16 cases (80.0％) died,and the overall survival rate was 20.0％ (4/20 cases) ,with survival time was (17.10 ± 2.24) months;of 134 MYCN non-amplification cases,96 cases (71.6％) survived and 38 cases (28.4％) died, with survival time of (28.71 ± 1.28)months.Survival analysis showed the cases with MYCN amplification had worse prognosis (x2 =19.596, P ＜ 0.05).Conclusions Patients with MYCN amplification had poorer prognosis and lower incidence of MYCN amplification of pediatric NT was found in China.

MYCN gene monitoring in children's malignant solid tumors / 国际儿科学杂志

Although the incidence of malignant solid tumors in children is not high,it is one of the major death causes in children.MYCN gene amplification is an independent poor prognosis factor of neuroblastoma.MYCN gene involves in neuroblastoma tumorgenesis and is the major evidence of treatment.MYCN gene amplification could be detected in other children's solid tumor.It is associated with unfavorable medulloblastoma pathology,poor outcome of Wilms' tumor and alveolar rhabdomyosarcomas.This review compares different MYCN gene detection methods,makes a summary about the clinical relationship between MYCN gene and children's solid tumor and investigates the possibility of increasing cure rate of malignant solid tumors through molecular specificity treatment.

Detection of gene amplification in MYCN, C-MYC, MYCL1, ERBB2, EGFR, AKT2, and human papilloma virus in samples from cervical smear normal cytology, intraepithelial cervical neoplasia (CIN I, II, III), and cervical cancer / Detección de amplificación en los genes MYCN, C-MYC, MYCL1, ERBB2, EGFR y AKT2 y del virus de papiloma humano en muestras de cepillado cervical en mujeres con citología normal y con neoplasia intraepitelial cervical (NIC) I, II, III y cáncer de cuello uterino

Introduction: Cervical cancer is the second most common cancer among women worldwide and the second cause of cancer mortality in women. It has been demonstrated that the process of cervical carcinogenesis displays genetic and environmental epigenetic components. Currently, research is focused on new prognosis markers like oncogene amplification.Objectives: To perform detection of MYCN, C-MYC, MYCL1, ERBB2, EGFR, and AKT2 amplification. Additionally, to detect human papillomavirus in samples from normal cytology smear, cervical intraepithelial neoplasia (CIN) I, II, and III and cervical cancer patients.Methods: Papillomavirus (HPV) genotyping by reverse line blot (RLB) performed and gene amplification by detection with real-time PCR with Taqman probes.Results: HPV was present in 4% of the patients with normal cytology, 48% in CIN I, 63.6% in CIN II, 64% in CIN III, and 70.8% in cervical cancer. Genes amplified in cervical cancer were MYCN (39.1%), ERBB2 (34.7%), and MYCL1 (30.4%); showed higher amplification in high-grade lesions and cervical cancer in relation to low-grade lesions and normal cytology with statistically significant differences. Besides the genes, C-MYC, EGFR, and AKT2 were amplified in samples from patients with cervical cancer by 12%, 18%, and 13%, respectively; we did not find statistical differences.Conclusion: Higher prevalence of gene amplification and HPV was found in high-grade cervical lesions and cervical cancer.

Introducción: El cáncer cervical es el segundo cáncer más importante en mujeres a nivel mundial y es la segunda causa de muerte por cáncer en mujeres. Se ha demostrado que el proceso de carcinogénesis cervical presenta componentes tanto genéticos como epigenéticos y medio ambientales. En la actualidad, hay gran interés en la búsqueda de marcadores moleculares asociados con la progresión de esta enfermedad, uno de los posibles mecanismos y que además está poco estudiado en cáncer cervical es la amplificación génica de algunos oncogenes como la familia MYC, EGFR y AKT entre otros.Objetivos: Detectar la amplificación génica de MYCN, C-MYC, MYCL1, ERBB2, EGFR y AKT2 además de la presencia del virus de papiloma humano en cepillados cervicales en mujeres con citología normal o con neoplasia intraepitelial cervical (NIC) I, II y III o con cáncer cervical.Métodos: Se genotipificó mediante reverse line blot (RLB) el virus de papiloma humano (VPH) y se determinó el estado de amplificación génica de los genes mencionados mediante PCR en tiempo real utilizando sondas taqman.Resultados: El VPH se encontró presente en 4% de las pacientes con citología normal, en 48% en NIC I, 63.6% en NIC II, 64% en NIC III y 70.8% en cáncer cervical. Los genes MYCN, MYCL1 y ERBB2 mostraron mayor amplificación en lesiones de alto grado y cáncer con diferencias estadísticamente significativas a las lesiones de bajo grado y citología normal, en 39.1%, 34.7% y 30.4% respectivamente. Además, se encontraron amplificados los genes C-MYC, EGFR y AKT2, en muestras de pacientes con cáncer cervical, en 12%, 18% y 13% respectivamente. Sin embargo, no se observaron diferencias estadísticamente significativas con respecto a las lesiones de alto y bajo grado y citología normal.Conclusión: En las lesiones de alto grado como en cáncer cervical, se encuentra mayor prevalencia del virus al igual que se detectan mayor cantidad de alteraciones genéticas como la amplificación génica.

The performance of semi-quantitative differential PCR is similar to that of real-time PCR for the detection of the MYCN gene in neuroblastomas

Amplification of the MYCN gene in neuroblastomas is a potent biological marker of highly aggressive tumors, which are invariably fatal unless sound clinical management is applied. To determine the usefulness of semi-quantitative differential PCR (SQ-PCR) for accurate quantification of MYCN gene copy number, we evaluated the analytical performance of this method by comparing the results obtained with it for 101 tumor samples of neuroblastoma to that obtained by absolute and relative real-time PCR. Similar results were obtained for 100 (99 percent) samples, no significant difference was detected between the median log10 MYCN copy number (1.53 by SQ-PCR versus 1.55 by absolute real-time PCR), and the results of the two assays correlated closely (r = 0.8, Pearson correlation; P < 0.001). In the comparison of SQ-PCR and relative real-time PCR, SQ-PCR versus relative real-time PCR concordant results were found in 100 (99 percent) samples, no significant difference was found in median log10 MYCN copy number (1.53 by SQ-PCR versus 1.27 by relative real-time PCR), and the results of the two assays correlated closely (r = 0.8, Pearson correlation; P < 0.001). These findings indicate that the performance of SQ-PCR was comparable to that of real-time PCR for the amplification and quantification of MYCN copy number. Thus, SQ-PCR can be reliably used as an alternative assay in laboratories without facilities for real-time PCR.

Effect on Cell Cycle Progression by N-Myc Knockdown in SK-N-BE(2) Neuroblastoma Cell Line and Cytotoxicity with STI-571 Compound / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: Neuroblastoma is a common tumor in childhood, and generally exhibits heterogeneity and a malignant progression. MYCN expression and amplification profiles frequently correlate with therapeutic prognosis. Although it has been reported that MYCN silencing causes differentiation and apoptosis in human neuroblastoma cells, MYCN expression influences the cytotoxic potential of chemotherapeutic drugs via the deregulation of the cell cycle. STI-571 may constitute a promising therapeutic agent against neuroblastoma, particularly in cases in which c-Kit is expressed preferentially in MYCN-amplified neuroblastoma. MATERIALS AND METHODS: To determine whether STI-571 exerts a synergistic effect on cytotoxicity with MYCN expression, we assessed apoptotic cell death and cell cycle distribution after 72 h of exposure to STI-571 with or with out treatment of SK-N-BE(2) neuroblastoma cells with MYCN siRNA. RESULTS: MYCN siRNA-treated SK-N-BE(2) cells did not affect apoptosis and cells were arrested in G0/G1 phase after STI-571 treatment. CONCLUSIONS: siRNA therapy targeted to MYCN may not be effective when administered in combination with STI-571 treatment in cases of neuroblastoma. Therefore, chemotherapeutic drugs that target S or G2-M phase may prove ineffective when applied to cells arrested in the G0/1 phase as the result of MYCN knockdown and STI-571 treatment.

Chromogenic in situ hybridization using routine tissue sections: MYCN in neuroblastoma.

Background: Amplification of the MYCN (myc myelocytomatosis viral related oncogene, neuroblastoma derived) gene in neuroblastoma is associated with a poor prognosis. Methods for estimating MYCN gene copy number that are based on pooled cells do not address copy number heterogeneity at the cell level and can underestimate or even miss amplification. MYCN copy number can be directly assessed by fluorescence in situ hybridization, but evaluation of tissue histology is difficult if not impossible. Objective: This paper reviews chromogenic in situ hybridization (CJSH) as it applies to the MYCN gene in neuroblastoma. We compare this technique to other methods for determining gene copy number and highlight the advantages of CISH. Methods: We have developed a chromogenic method for in situ hybridization (CISH) that enables us to determine MYCN copy number on an individual cell basis. This technique uses light microscopy on routine paraffin sections, and therefore allows simultaneous assessment of tumour histology. Results: In a previous study, CISH identified 100 % of the cases that were known to be amplified by other techniques and proved to be more sensitive than Southern blotting or the quantitative DNA polymerase chain reaction. The MYCN copy number is generally believed not to vary within a tumour, nor between tumour samples, including primary vs. metastases, and pre-and post-treatment specimens. However, we found heterogeneity from cell to cell, with ~30 % of amplified tumours showing >50 % variation in MYCN copy between cells. Conclusion: For detection of gene amplification, CISH has all the advantages of FISH but in addition, needs no special microscopy or image capturing systems, and preparations are permanent. In the case of neuroblastoma, CISH has disclosed considerable heterogeneity in MYCN copy number between cells in a tumour. Heterogeneity reflects different tumour clones and its role has been under-recognized in neuroblastoma biology. Additional studies are needed to investigate the significance of tumour heterogeneity in neuroblastoma, and whether the aggressive (i.e., MYCN-amplified) clones are more likely to metastasize, survive treatment modalities, and ultimately kill the patient.