title: Clinical significance of a highly sensitive analysis for gene dosage and the expression level of MYCN in neuroblastoma.
abstract: BACKGROUND
  The amplification of the MYCN gene is one of the most powerful adverse prognosis factors in neuroblastoma, but the clinical significance of an enhanced expression of MYCN remains controversial. To reassess the clinical implications of MYCN amplification and expression in neuroblastoma, the status of amplification and the expression level of the MYCN gene of primary neuroblastoma samples were analyzed using highly sensitive analyses.
  
  
  METHODS
  Using a quantitative polymerase chain reaction (PCR) method (TaqMan), the gene dosages (MYCN/p53) of 66 primary neuroblastoma samples were determined. In all 66 samples, the status of MYCN amplification has been determined previously by the Southern blotting method. Of the 54 samples with a single copy of MYCN based on the Southern blotting method, 23 samples were analyzed for MYCN amplification using the fluorescence in situ hybridization (FISH) method. The expression levels (MYCN/GAPDH) of 56 samples were determined by a quantitative reverse transcriptase (RT)-PCR method.
  
  
  RESULTS
  Of the 54 samples with a single copy of MYCN based on the Southern blotting method, 46 samples showed MYCN gene dosages of less than 2.0, whereas the remaining 8 samples with dosages of more than 2.0 were tumors from patients with advanced-stage disease. The results of FISH supported the fact that these 8 samples contained a small number of MYCN-amplified cells. The cases of MYCN gene dosages of more than 2.0 were significantly associated with all other unfavorable prognostic factors (an age of >1 year at diagnosis [P <.0001], nonmass screening [P =.0003], advanced stage [P <.0001], diploid or tetraploid [P <.0001], and a Shimada unfavorable histology [P <.0001]). MYCN gene dosages of more than 2.0 were significantly associated with a high expression of MYCN (P =.0459). However, the expression level of MYCN was not significantly associated with any other prognostic factors.
  
  
  CONCLUSIONS
  Quantitative PCR may thus be a useful modality for performing a highly sensitive and accurate assessment of the amplification and expression levels of the MYCN gene. In particular, the combination of the quantitative PCR system and the FISH method is considered to be a highly effective method for evaluating the status of MYCN amplification. In this highly sensitive analysis, MYCN amplification (MYCN/p53 > or = 2.0) was reconfirmed to be a strongly unfavorable factor, whereas the expression level of MYCN does not appear to be an independently significant prognosis factor.
authors.affiliation: Department of Pediatric Surgery, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
authors.forename: Shinji
authors.initials: S
authors.lastname: Tanaka
authors.forename: Tatsuro
authors.initials: T
authors.lastname: Tajiri
authors.forename: Shin-ichi
authors.initials: S
authors.lastname: Noguchi
authors.forename: Kumiko
authors.initials: K
authors.lastname: Shono
authors.forename: Kenji
authors.initials: K
authors.lastname: Ihara
authors.forename: Toshiro
authors.initials: T
authors.lastname: Hara
authors.forename: Sachiyo
authors.initials: S
authors.lastname: Suita
mesh_terms: D002648:Child; D002675:Child, Preschool; D004247:DNA / Q000302:isolation & purification; D005260:Female; D005784:Gene Amplification; D018628:Gene Dosage; D015870:Gene Expression; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D007223:Infant; D008297:Male; D000071447:N-Myc Proto-Oncogene Protein; D009447:Neuroblastoma / Q000235:genetics* / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D015513:Oncogene Proteins / Q000235:genetics* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011379:Prognosis; D012313:RNA / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: C459801:MYCN protein, human; D000071447:N-Myc Proto-Oncogene Protein; D009687:Nuclear Proteins; D015513:Oncogene Proteins; D012313:RNA; D004247:DNA