A multilocus technique for risk evaluation of patients with neuroblastoma

Inge M. Ambros, Bettina Brunner, Gerhard Aigner, Clare Bedwell, Klaus Beiske, Jean Bénard, Nick Bown, Valerie Combaret, Jerome Couturier, Raffaella Defferrari, Nicole Gross, Marta Jeison, John Lunec, Barbara Marques, Tommy Martinsson, Katia Mazzocco, Rosa Noguera, Gudrun Schleiermacher, Frank Speleman, Ray StallingsGian Paolo Tonini, Deborah A. Tweddle, Alexander Valent, Ales Vicha, Nadine Van Roy, Eva Villamon, Andrea Ziegler, Sandra Preuner, Mario Drobics, Ruth Ladenstein, Gabriele Amann, Robert J L Schuit, Ulrike Pötschger, Peter F. Ambros

Research output: Contribution to journalArticlepeer-review


Purpose: 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. Experimental Design: 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.

Original languageEnglish
Pages (from-to)792-804
Number of pages13
JournalClinical Cancer Research
Issue number4
Publication statusPublished - Feb 15 2011

ASJC Scopus subject areas

  • Cancer Research
  • Oncology


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