title: A multilocus technique for risk evaluation of patients with neuroblastoma.
abstract: OBJECTIVE
  Precise and comprehensive analysis of neuroblastoma genetics is essential for accurate risk evaluation and only pangenomic/multilocus approaches fulfill the present-day requirements. We present the establishment and validation of the PCR-based multiplex ligation-dependent probe amplification (MLPA) technique for neuroblastoma.
  
  
  METHODS
  A neuroblastoma-specific MLPA kit was designed by the SIOP Europe Neuroblastoma Biology Committee in cooperation with MRC-Holland. The contained target sequences cover 19 chromosomal arms and reference loci. Validation was performed by single locus and pangenomic techniques (n = 174). Dilution experiments for determination of minimal tumor cell percentage were performed and testing of reproducibility was checked by interlaboratory testing (n = 15). Further 156 neuroblastomas were used for establishing the amplification cutoff level.
  
  
  RESULTS
  The MLPA technique was tested in 310 neuroblastomas and 8 neuroblastoma cell lines (including validation and amplification cutoff level testing). Intertechnique validation showed a high concordance rate (99.5%). Interlaboratory MLPA testing (κ = 0.95, P < 0.01) revealed 7 discrepant of 1,490 results (0.5%). Validation by pangenomic techniques showed a single discordance of 190 consensus results (0.5%). The test results led to formulation of interpretation standards and to a kit revision. The minimal tumor cell percentage was fixed at 60%.
  
  
  CONCLUSIONS
  The recently designed neuroblastoma-specific MLPA kit covers all chromosomal regions demanded by the International Neuroblastoma Risk Group for therapy stratification and includes all hitherto described genetic loci of prognostic interest for future studies and can be modified or extended at any time. Moreover, the technique is cost effective, reliable, and robust with a high interlaboratory and intertechnique concordance.
authors.affiliation: CCRI, Children's Cancer Research Institute, St. Anna Kinderkrebsforschung, Vienna, Austria. ambros@ccri.at
authors.forename: Inge M
authors.initials: IM
authors.lastname: Ambros
authors.forename: Bettina
authors.initials: B
authors.lastname: Brunner
authors.forename: Gerhard
authors.initials: G
authors.lastname: Aigner
authors.forename: Clare
authors.initials: C
authors.lastname: Bedwell
authors.forename: Klaus
authors.initials: K
authors.lastname: Beiske
authors.forename: Jean
authors.initials: J
authors.lastname: Bénard
authors.forename: Nick
authors.initials: N
authors.lastname: Bown
authors.forename: Valerie
authors.initials: V
authors.lastname: Combaret
authors.forename: Jerome
authors.initials: J
authors.lastname: Couturier
authors.forename: Raffaella
authors.initials: R
authors.lastname: Defferrari
authors.forename: Nicole
authors.initials: N
authors.lastname: Gross
authors.forename: Marta
authors.initials: M
authors.lastname: Jeison
authors.forename: John
authors.initials: J
authors.lastname: Lunec
authors.forename: Barbara
authors.initials: B
authors.lastname: Marques
authors.forename: Tommy
authors.initials: T
authors.lastname: Martinsson
authors.forename: Katia
authors.initials: K
authors.lastname: Mazzocco
authors.forename: Rosa
authors.initials: R
authors.lastname: Noguera
authors.forename: Gudrun
authors.initials: G
authors.lastname: Schleiermacher
authors.forename: Frank
authors.initials: F
authors.lastname: Speleman
authors.forename: Ray
authors.initials: R
authors.lastname: Stallings
authors.forename: Gian Paolo
authors.initials: GP
authors.lastname: Tonini
authors.forename: Deborah A
authors.initials: DA
authors.lastname: Tweddle
authors.forename: Alexander
authors.initials: A
authors.lastname: Valent
authors.forename: Ales
authors.initials: A
authors.lastname: Vicha
authors.forename: Nadine Van
authors.initials: NV
authors.lastname: Roy
authors.forename: Eva
authors.initials: E
authors.lastname: Villamon
authors.forename: Andrea
authors.initials: A
authors.lastname: Ziegler
authors.forename: Sandra
authors.initials: S
authors.lastname: Preuner
authors.forename: Mario
authors.initials: M
authors.lastname: Drobics
authors.forename: Ruth
authors.initials: R
authors.lastname: Ladenstein
authors.forename: Gabriele
authors.initials: G
authors.lastname: Amann
authors.forename: Robert J L
authors.initials: RJ
authors.lastname: Schuit
authors.forename: Ulrike
authors.initials: U
authors.lastname: Pötschger
authors.forename: Peter F
authors.initials: PF
authors.lastname: Ambros
mesh_terms: D003196:Computer Graphics; D005784:Gene Amplification; D056426:Genetic Loci; D005819:Genetic Markers; D006801:Humans; D057230:Limit of Detection; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D009154:Mutation; D000071447:N-Myc Proto-Oncogene Protein; D009447:Neuroblastoma / Q000235:genetics* / Q000473:pathology; D009687:Nuclear Proteins / Q000235:genetics; D015513:Oncogene Proteins / Q000235:genetics; D018570:Risk Assessment
chemical_list: D005819:Genetic Markers; C459801:MYCN protein, human; D000071447:N-Myc Proto-Oncogene Protein; D009687:Nuclear Proteins; D015513:Oncogene Proteins