Validation of a MEK/MET-specific NGS panel for early phase trial interrogation

P. Maxwell, jurgen del favero, Marc-Aurel Fuchs, Josef Tabernero, Tim Maughan, Mark Middleton, Richard Adams, Christian Rolfo, Bryan T Hennessy, Pierre Laurent=puig, alberto Bardelli, Thierry Andre, Vlad Popovici, Patrick Johnston, Richard Wilson, Mark Lawler, Sandra Van Schaeybroeck, Manuel Salto-Tellez

    Research output: Chapter in Book/Report/Conference proceedingConference contribution

    Abstract

    Introduction - MErCuRIC is a Phase Ib/II clinical trial study using a combined MEK1/2 - cMET inhibition in RAS MT and RAS WT (with aberrant c[[unable to display character: ‐]]MET) colorectal cancer patients. As part of the discovery efforts on the cases enrolled in Phase I, we aimed to analyze the mutation status of 10 genes that could potentially be associated to the doublet MEK/MET inhibition. This study compared the MEK/MET-specific panel with the Ion Torrent 50 gene panel, aiming to compare: long-established, commercially available panels against this newly developed panel; the Ion Torrent PGM2 platform against Illumina MiSeq v.3 600 bp chemistry; hot-spot-based against full exomes-designed; and to compare the use of different bioinformatics reporter systems. The overlapping genes between the panels were: EGFR (n=3); BRAF (n=4); KRAS (n=8); NRAS (n=1); MET (n=8); PIK3CA (n=6); and ERBB2 (n=5). Summary of Method NGS Design - The Multiplicom - MErCuRIC specific MASTR assay includes PTEN, MAP2K1 (MEK), EGFR, KRAS, NRAS, BRAF, PIK3CA, ERBB2, MET and PIK3R1, involving 257 amplicons in 4 plexes covering all coding exons of the 10 genes. Of the 257, 21 are control amplicons to allow for gene deletions/amplifications. The average length of the amplicons is 198 bp ranging from 124 bp to 255 bp. Validation - From a pool of 120 routine tumour samples characterised with a 50 gene mutation panel (Ion Torrent PGM2) and confirmed by Sanger sequencing and/or COBAS (Roche) QPCR, 24 FFPE cases were selected representing colorectal cancer and 4 other solid tumour types; all included a variety of DNA quality, and DNA concentration was standardized prior to library preparation. Pre-analytical handling was in accordance with established protocols in a laboratory, clinically-accredited in the UK for tissue-based, anatomical pathology testing. Results The evaluation of this MEK/MET-specific panel (Illumina MiSeq platform) resulted in 100% coverage of all targets, a higher than 97% on target reads and higher than 99% of all amplicons within 20% of mean coverage. The design minimized the areas of low coverage. A small part of exon 9 of ERBB2 was covered suboptimally: the low covered region is 30 bp in size located at the 5’ end of exon 9. It is unlikely that this low coverage led to false negative results since no mutations are reported in the COSMIC database for this DNA fragment. The results were concordant in relation to mutations involved in the genes stated above. Importantly, the percentages of allele frequency between both methods were similar, with variations ranging from 0.2% to 11.5% with an average variation of 5.2%. Insertion/deletion (Indel) detection however, required alternative bioinformatics pathways. Conclusion After combining well-established quality metrics to cover pre-analytical aspects with suitable technologies such as the MiSeq platform (Illumina) and appropriate bioinformatics, we recognize that this MEK/MET-specific NGS panel is fit for purpose.
    Original languageEnglish
    Title of host publicationValidation of a MEK/MET-specific NGS panel for early phase trial interrogation
    PublisherProceedings: AACR Annual Meeting 2016; April 16-20 New Orleans
    Volumeabstract 1396
    Publication statusPublished - 01 May 2016

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    Mitogen-Activated Protein Kinase Kinases
    Computational Biology
    Exons
    Mutation
    Genes
    Ions
    Colorectal Neoplasms
    Overlapping Genes
    Exome
    Phase II Clinical Trials
    Gene Amplification
    DNA
    Nucleic Acid Databases
    Gene Deletion
    Gene Frequency
    Libraries
    Neoplasms
    Pathology
    Technology

    Cite this

    Maxwell, P., del favero, J., Fuchs, M-A., Tabernero, J., Maughan, T., Middleton, M., ... Salto-Tellez, M. (2016). Validation of a MEK/MET-specific NGS panel for early phase trial interrogation. In Validation of a MEK/MET-specific NGS panel for early phase trial interrogation (Vol. abstract 1396). Proceedings: AACR Annual Meeting 2016; April 16-20 New Orleans.
    Maxwell, P. ; del favero, jurgen ; Fuchs, Marc-Aurel ; Tabernero, Josef ; Maughan, Tim ; Middleton, Mark ; Adams, Richard ; Rolfo, Christian ; Hennessy, Bryan T ; Laurent=puig, Pierre ; Bardelli, alberto ; Andre, Thierry ; Popovici, Vlad ; Johnston, Patrick ; Wilson, Richard ; Lawler, Mark ; Van Schaeybroeck, Sandra ; Salto-Tellez, Manuel. / Validation of a MEK/MET-specific NGS panel for early phase trial interrogation. Validation of a MEK/MET-specific NGS panel for early phase trial interrogation . Vol. abstract 1396 Proceedings: AACR Annual Meeting 2016; April 16-20 New Orleans, 2016.
    @inproceedings{92762238b33044d1969d6fd12053c6bb,
    title = "Validation of a MEK/MET-specific NGS panel for early phase trial interrogation",
    abstract = "Introduction - MErCuRIC is a Phase Ib/II clinical trial study using a combined MEK1/2 - cMET inhibition in RAS MT and RAS WT (with aberrant c[[unable to display character: ‐]]MET) colorectal cancer patients. As part of the discovery efforts on the cases enrolled in Phase I, we aimed to analyze the mutation status of 10 genes that could potentially be associated to the doublet MEK/MET inhibition. This study compared the MEK/MET-specific panel with the Ion Torrent 50 gene panel, aiming to compare: long-established, commercially available panels against this newly developed panel; the Ion Torrent PGM2 platform against Illumina MiSeq v.3 600 bp chemistry; hot-spot-based against full exomes-designed; and to compare the use of different bioinformatics reporter systems. The overlapping genes between the panels were: EGFR (n=3); BRAF (n=4); KRAS (n=8); NRAS (n=1); MET (n=8); PIK3CA (n=6); and ERBB2 (n=5). Summary of Method NGS Design - The Multiplicom - MErCuRIC specific MASTR assay includes PTEN, MAP2K1 (MEK), EGFR, KRAS, NRAS, BRAF, PIK3CA, ERBB2, MET and PIK3R1, involving 257 amplicons in 4 plexes covering all coding exons of the 10 genes. Of the 257, 21 are control amplicons to allow for gene deletions/amplifications. The average length of the amplicons is 198 bp ranging from 124 bp to 255 bp. Validation - From a pool of 120 routine tumour samples characterised with a 50 gene mutation panel (Ion Torrent PGM2) and confirmed by Sanger sequencing and/or COBAS (Roche) QPCR, 24 FFPE cases were selected representing colorectal cancer and 4 other solid tumour types; all included a variety of DNA quality, and DNA concentration was standardized prior to library preparation. Pre-analytical handling was in accordance with established protocols in a laboratory, clinically-accredited in the UK for tissue-based, anatomical pathology testing. Results The evaluation of this MEK/MET-specific panel (Illumina MiSeq platform) resulted in 100{\%} coverage of all targets, a higher than 97{\%} on target reads and higher than 99{\%} of all amplicons within 20{\%} of mean coverage. The design minimized the areas of low coverage. A small part of exon 9 of ERBB2 was covered suboptimally: the low covered region is 30 bp in size located at the 5’ end of exon 9. It is unlikely that this low coverage led to false negative results since no mutations are reported in the COSMIC database for this DNA fragment. The results were concordant in relation to mutations involved in the genes stated above. Importantly, the percentages of allele frequency between both methods were similar, with variations ranging from 0.2{\%} to 11.5{\%} with an average variation of 5.2{\%}. Insertion/deletion (Indel) detection however, required alternative bioinformatics pathways. Conclusion After combining well-established quality metrics to cover pre-analytical aspects with suitable technologies such as the MiSeq platform (Illumina) and appropriate bioinformatics, we recognize that this MEK/MET-specific NGS panel is fit for purpose.",
    author = "P. Maxwell and {del favero}, jurgen and Marc-Aurel Fuchs and Josef Tabernero and Tim Maughan and Mark Middleton and Richard Adams and Christian Rolfo and Hennessy, {Bryan T} and Pierre Laurent=puig and alberto Bardelli and Thierry Andre and Vlad Popovici and Patrick Johnston and Richard Wilson and Mark Lawler and {Van Schaeybroeck}, Sandra and Manuel Salto-Tellez",
    year = "2016",
    month = "5",
    day = "1",
    language = "English",
    volume = "abstract 1396",
    booktitle = "Validation of a MEK/MET-specific NGS panel for early phase trial interrogation",
    publisher = "Proceedings: AACR Annual Meeting 2016; April 16-20 New Orleans",

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    Maxwell, P, del favero, J, Fuchs, M-A, Tabernero, J, Maughan, T, Middleton, M, Adams, R, Rolfo, C, Hennessy, BT, Laurent=puig, P, Bardelli, A, Andre, T, Popovici, V, Johnston, P, Wilson, R, Lawler, M, Van Schaeybroeck, S & Salto-Tellez, M 2016, Validation of a MEK/MET-specific NGS panel for early phase trial interrogation. in Validation of a MEK/MET-specific NGS panel for early phase trial interrogation . vol. abstract 1396, Proceedings: AACR Annual Meeting 2016; April 16-20 New Orleans.

    Validation of a MEK/MET-specific NGS panel for early phase trial interrogation. / Maxwell, P.; del favero, jurgen; Fuchs, Marc-Aurel; Tabernero, Josef; Maughan, Tim; Middleton, Mark; Adams, Richard; Rolfo, Christian; Hennessy, Bryan T; Laurent=puig, Pierre; Bardelli, alberto; Andre, Thierry; Popovici, Vlad; Johnston, Patrick; Wilson, Richard; Lawler, Mark; Van Schaeybroeck, Sandra; Salto-Tellez, Manuel.

    Validation of a MEK/MET-specific NGS panel for early phase trial interrogation . Vol. abstract 1396 Proceedings: AACR Annual Meeting 2016; April 16-20 New Orleans, 2016.

    Research output: Chapter in Book/Report/Conference proceedingConference contribution

    TY - GEN

    T1 - Validation of a MEK/MET-specific NGS panel for early phase trial interrogation

    AU - Maxwell, P.

    AU - del favero, jurgen

    AU - Fuchs, Marc-Aurel

    AU - Tabernero, Josef

    AU - Maughan, Tim

    AU - Middleton, Mark

    AU - Adams, Richard

    AU - Rolfo, Christian

    AU - Hennessy, Bryan T

    AU - Laurent=puig, Pierre

    AU - Bardelli, alberto

    AU - Andre, Thierry

    AU - Popovici, Vlad

    AU - Johnston, Patrick

    AU - Wilson, Richard

    AU - Lawler, Mark

    AU - Van Schaeybroeck, Sandra

    AU - Salto-Tellez, Manuel

    PY - 2016/5/1

    Y1 - 2016/5/1

    N2 - Introduction - MErCuRIC is a Phase Ib/II clinical trial study using a combined MEK1/2 - cMET inhibition in RAS MT and RAS WT (with aberrant c[[unable to display character: ‐]]MET) colorectal cancer patients. As part of the discovery efforts on the cases enrolled in Phase I, we aimed to analyze the mutation status of 10 genes that could potentially be associated to the doublet MEK/MET inhibition. This study compared the MEK/MET-specific panel with the Ion Torrent 50 gene panel, aiming to compare: long-established, commercially available panels against this newly developed panel; the Ion Torrent PGM2 platform against Illumina MiSeq v.3 600 bp chemistry; hot-spot-based against full exomes-designed; and to compare the use of different bioinformatics reporter systems. The overlapping genes between the panels were: EGFR (n=3); BRAF (n=4); KRAS (n=8); NRAS (n=1); MET (n=8); PIK3CA (n=6); and ERBB2 (n=5). Summary of Method NGS Design - The Multiplicom - MErCuRIC specific MASTR assay includes PTEN, MAP2K1 (MEK), EGFR, KRAS, NRAS, BRAF, PIK3CA, ERBB2, MET and PIK3R1, involving 257 amplicons in 4 plexes covering all coding exons of the 10 genes. Of the 257, 21 are control amplicons to allow for gene deletions/amplifications. The average length of the amplicons is 198 bp ranging from 124 bp to 255 bp. Validation - From a pool of 120 routine tumour samples characterised with a 50 gene mutation panel (Ion Torrent PGM2) and confirmed by Sanger sequencing and/or COBAS (Roche) QPCR, 24 FFPE cases were selected representing colorectal cancer and 4 other solid tumour types; all included a variety of DNA quality, and DNA concentration was standardized prior to library preparation. Pre-analytical handling was in accordance with established protocols in a laboratory, clinically-accredited in the UK for tissue-based, anatomical pathology testing. Results The evaluation of this MEK/MET-specific panel (Illumina MiSeq platform) resulted in 100% coverage of all targets, a higher than 97% on target reads and higher than 99% of all amplicons within 20% of mean coverage. The design minimized the areas of low coverage. A small part of exon 9 of ERBB2 was covered suboptimally: the low covered region is 30 bp in size located at the 5’ end of exon 9. It is unlikely that this low coverage led to false negative results since no mutations are reported in the COSMIC database for this DNA fragment. The results were concordant in relation to mutations involved in the genes stated above. Importantly, the percentages of allele frequency between both methods were similar, with variations ranging from 0.2% to 11.5% with an average variation of 5.2%. Insertion/deletion (Indel) detection however, required alternative bioinformatics pathways. Conclusion After combining well-established quality metrics to cover pre-analytical aspects with suitable technologies such as the MiSeq platform (Illumina) and appropriate bioinformatics, we recognize that this MEK/MET-specific NGS panel is fit for purpose.

    AB - Introduction - MErCuRIC is a Phase Ib/II clinical trial study using a combined MEK1/2 - cMET inhibition in RAS MT and RAS WT (with aberrant c[[unable to display character: ‐]]MET) colorectal cancer patients. As part of the discovery efforts on the cases enrolled in Phase I, we aimed to analyze the mutation status of 10 genes that could potentially be associated to the doublet MEK/MET inhibition. This study compared the MEK/MET-specific panel with the Ion Torrent 50 gene panel, aiming to compare: long-established, commercially available panels against this newly developed panel; the Ion Torrent PGM2 platform against Illumina MiSeq v.3 600 bp chemistry; hot-spot-based against full exomes-designed; and to compare the use of different bioinformatics reporter systems. The overlapping genes between the panels were: EGFR (n=3); BRAF (n=4); KRAS (n=8); NRAS (n=1); MET (n=8); PIK3CA (n=6); and ERBB2 (n=5). Summary of Method NGS Design - The Multiplicom - MErCuRIC specific MASTR assay includes PTEN, MAP2K1 (MEK), EGFR, KRAS, NRAS, BRAF, PIK3CA, ERBB2, MET and PIK3R1, involving 257 amplicons in 4 plexes covering all coding exons of the 10 genes. Of the 257, 21 are control amplicons to allow for gene deletions/amplifications. The average length of the amplicons is 198 bp ranging from 124 bp to 255 bp. Validation - From a pool of 120 routine tumour samples characterised with a 50 gene mutation panel (Ion Torrent PGM2) and confirmed by Sanger sequencing and/or COBAS (Roche) QPCR, 24 FFPE cases were selected representing colorectal cancer and 4 other solid tumour types; all included a variety of DNA quality, and DNA concentration was standardized prior to library preparation. Pre-analytical handling was in accordance with established protocols in a laboratory, clinically-accredited in the UK for tissue-based, anatomical pathology testing. Results The evaluation of this MEK/MET-specific panel (Illumina MiSeq platform) resulted in 100% coverage of all targets, a higher than 97% on target reads and higher than 99% of all amplicons within 20% of mean coverage. The design minimized the areas of low coverage. A small part of exon 9 of ERBB2 was covered suboptimally: the low covered region is 30 bp in size located at the 5’ end of exon 9. It is unlikely that this low coverage led to false negative results since no mutations are reported in the COSMIC database for this DNA fragment. The results were concordant in relation to mutations involved in the genes stated above. Importantly, the percentages of allele frequency between both methods were similar, with variations ranging from 0.2% to 11.5% with an average variation of 5.2%. Insertion/deletion (Indel) detection however, required alternative bioinformatics pathways. Conclusion After combining well-established quality metrics to cover pre-analytical aspects with suitable technologies such as the MiSeq platform (Illumina) and appropriate bioinformatics, we recognize that this MEK/MET-specific NGS panel is fit for purpose.

    M3 - Conference contribution

    VL - abstract 1396

    BT - Validation of a MEK/MET-specific NGS panel for early phase trial interrogation

    PB - Proceedings: AACR Annual Meeting 2016; April 16-20 New Orleans

    ER -

    Maxwell P, del favero J, Fuchs M-A, Tabernero J, Maughan T, Middleton M et al. Validation of a MEK/MET-specific NGS panel for early phase trial interrogation. In Validation of a MEK/MET-specific NGS panel for early phase trial interrogation . Vol. abstract 1396. Proceedings: AACR Annual Meeting 2016; April 16-20 New Orleans. 2016