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 journalArticle

35 Citations (Scopus)

Abstract

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
Volume17
Issue number4
DOIs
Publication statusPublished - Feb 15 2011

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Neuroblastoma
Multiplex Polymerase Chain Reaction
Genetic Loci
Group Psychotherapy
Netherlands
Neoplasms
Consensus
Research Design
Costs and Cost Analysis
Cell Line
Polymerase Chain Reaction

ASJC Scopus subject areas

  • Cancer Research
  • Oncology

Cite this

Ambros, I. M., Brunner, B., Aigner, G., Bedwell, C., Beiske, K., Bénard, J., ... Ambros, P. F. (2011). A multilocus technique for risk evaluation of patients with neuroblastoma. Clinical Cancer Research, 17(4), 792-804. https://doi.org/10.1158/1078-0432.CCR-10-0830

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

In: Clinical Cancer Research, Vol. 17, No. 4, 15.02.2011, p. 792-804.

Research output: Contribution to journalArticle

Ambros, IM, Brunner, B, Aigner, G, Bedwell, C, Beiske, K, Bénard, J, Bown, N, Combaret, V, Couturier, J, Defferrari, R, Gross, N, Jeison, M, Lunec, J, Marques, B, Martinsson, T, Mazzocco, K, Noguera, R, Schleiermacher, G, Speleman, F, Stallings, R, Tonini, GP, Tweddle, DA, Valent, A, Vicha, A, Van Roy, N, Villamon, E, Ziegler, A, Preuner, S, Drobics, M, Ladenstein, R, Amann, G, Schuit, RJL, Pötschger, U & Ambros, PF 2011, 'A multilocus technique for risk evaluation of patients with neuroblastoma', Clinical Cancer Research, vol. 17, no. 4, pp. 792-804. https://doi.org/10.1158/1078-0432.CCR-10-0830
Ambros IM, Brunner B, Aigner G, Bedwell C, Beiske K, Bénard J et al. A multilocus technique for risk evaluation of patients with neuroblastoma. Clinical Cancer Research. 2011 Feb 15;17(4):792-804. https://doi.org/10.1158/1078-0432.CCR-10-0830
Ambros, Inge M. ; Brunner, Bettina ; Aigner, Gerhard ; Bedwell, Clare ; Beiske, Klaus ; Bénard, Jean ; Bown, Nick ; Combaret, Valerie ; Couturier, Jerome ; Defferrari, Raffaella ; Gross, Nicole ; Jeison, Marta ; Lunec, John ; Marques, Barbara ; Martinsson, Tommy ; Mazzocco, Katia ; Noguera, Rosa ; Schleiermacher, Gudrun ; Speleman, Frank ; Stallings, Ray ; Tonini, Gian Paolo ; Tweddle, Deborah A. ; Valent, Alexander ; Vicha, Ales ; Van Roy, Nadine ; Villamon, Eva ; Ziegler, Andrea ; Preuner, Sandra ; Drobics, Mario ; Ladenstein, Ruth ; Amann, Gabriele ; Schuit, Robert J L ; Pötschger, Ulrike ; Ambros, Peter F. / A multilocus technique for risk evaluation of patients with neuroblastoma. In: Clinical Cancer Research. 2011 ; Vol. 17, No. 4. pp. 792-804.
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AU - Ambros, Inge M.

AU - Brunner, Bettina

AU - Aigner, Gerhard

AU - Bedwell, Clare

AU - Beiske, Klaus

AU - Bénard, Jean

AU - Bown, Nick

AU - Combaret, Valerie

AU - Couturier, Jerome

AU - Defferrari, Raffaella

AU - Gross, Nicole

AU - Jeison, Marta

AU - Lunec, John

AU - Marques, Barbara

AU - Martinsson, Tommy

AU - Mazzocco, Katia

AU - Noguera, Rosa

AU - Schleiermacher, Gudrun

AU - Speleman, Frank

AU - Stallings, Ray

AU - Tonini, Gian Paolo

AU - Tweddle, Deborah A.

AU - Valent, Alexander

AU - Vicha, Ales

AU - Van Roy, Nadine

AU - Villamon, Eva

AU - Ziegler, Andrea

AU - Preuner, Sandra

AU - Drobics, Mario

AU - Ladenstein, Ruth

AU - Amann, Gabriele

AU - Schuit, Robert J L

AU - Pötschger, Ulrike

AU - Ambros, Peter F.

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N2 - 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.

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