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Validation and utilisation of high-coverage next-generation sequencing to deliver the pharmacological audit trail.

Ong M, Carreira S, Goodall J, Mateo J, Figueiredo I, Rodrigues DN, Perkins G, Seed G, Yap TA, Attard G, de Bono JS - Br. J. Cancer (2014)

Bottom Line: Predictive biomarker development is a key challenge for novel cancer therapeutics.We found 97% concordance of mutation calls by MiSeq and IT-PGM at a variant allele frequency ⩾13% and ⩾500 × depth coverage, and 91% concordance between MiSeq and Sequenom.Common 'actionable' mutations involved deoxyribonucleic acid (DNA) repair (51%), RAS-RAF-MEK (35%), Wnt (26%), and PI3K-AKT-mTOR (24%) signalling.

View Article: PubMed Central - PubMed

Affiliation: 1] Cancer Biomarkers Team, The Institute of Cancer Research, 15 Cotswold Road, Sutton, Surrey SM2 5NG, UK [2] Drug Development Unit, The Royal Marsden NHS Foundation Trust, Sutton, Surrey SM2 5PT, UK.

ABSTRACT

Background: Predictive biomarker development is a key challenge for novel cancer therapeutics. We explored the feasibility of next-generation sequencing (NGS) to validate exploratory genomic biomarkers that impact phase I trial selection.

Methods: We prospectively enrolled 158 patients with advanced solid tumours referred for phase I clinical trials at the Royal Marsden Hospital (October 2012 to March 2013). After fresh and/or archived tumour tissue were obtained, 93 patients remained candidates for phase I trials. Results from tumour sequencing on the Illumina MiSeq were cross-validated in 27 out of 93 patients on the Ion Torrent Personal Genome Machine (IT-PGM) blinded to results. MiSeq validation with Sequenom MassARRAY OncoCarta 1.0 (Sequenom Inc., San Diego, CA, USA) was performed in a separate cohort.

Results: We found 97% concordance of mutation calls by MiSeq and IT-PGM at a variant allele frequency ⩾13% and ⩾500 × depth coverage, and 91% concordance between MiSeq and Sequenom. Common 'actionable' mutations involved deoxyribonucleic acid (DNA) repair (51%), RAS-RAF-MEK (35%), Wnt (26%), and PI3K-AKT-mTOR (24%) signalling. Out of 53, 29 (55%) patients participating in phase I trials were recommended based on identified actionable mutations.

Conclusions: Targeted high-coverage NGS panels are a highly feasible single-centre technology well-suited to cross-platform validation, enrichment of trials with molecularly defined populations and hypothesis testing early in drug development.

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Related in: MedlinePlus

Patient and sample flow diagram. Abbreviations: IT-PGM=Ion Torrent Personal Genome Machine; NGS=next-generation sequencing.
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fig1: Patient and sample flow diagram. Abbreviations: IT-PGM=Ion Torrent Personal Genome Machine; NGS=next-generation sequencing.

Mentions: The Ion AmpliSeq Cancer Hotspot Panel v2 contains a single pool of primers used to perform multiplexed PCR for preparation of amplicon libraries covering 207 ROIs in 50 cancer-related genes (Supplementary Tables 1 and 2, Figure 1A and B), in which 2175 mutations are represented at least twice in COSMIC. Twenty-seven randomly selected samples from the prospective cohort were selected to compare MiSeq and PGM sequencing. Targeted sequencing was performed following the manufacturer's protocol (see Supplementary Methods). Bioinformatic analyses were performed using the PGM Torrent Suite software (version 3·4·2) including a Torrent Variant Caller for single-nucleotide polymorphisms and indel variants across a reference BED file, and Nextgene (from Biogene) for comparison with the MiSeq data.


Validation and utilisation of high-coverage next-generation sequencing to deliver the pharmacological audit trail.

Ong M, Carreira S, Goodall J, Mateo J, Figueiredo I, Rodrigues DN, Perkins G, Seed G, Yap TA, Attard G, de Bono JS - Br. J. Cancer (2014)

Patient and sample flow diagram. Abbreviations: IT-PGM=Ion Torrent Personal Genome Machine; NGS=next-generation sequencing.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4150267&req=5

fig1: Patient and sample flow diagram. Abbreviations: IT-PGM=Ion Torrent Personal Genome Machine; NGS=next-generation sequencing.
Mentions: The Ion AmpliSeq Cancer Hotspot Panel v2 contains a single pool of primers used to perform multiplexed PCR for preparation of amplicon libraries covering 207 ROIs in 50 cancer-related genes (Supplementary Tables 1 and 2, Figure 1A and B), in which 2175 mutations are represented at least twice in COSMIC. Twenty-seven randomly selected samples from the prospective cohort were selected to compare MiSeq and PGM sequencing. Targeted sequencing was performed following the manufacturer's protocol (see Supplementary Methods). Bioinformatic analyses were performed using the PGM Torrent Suite software (version 3·4·2) including a Torrent Variant Caller for single-nucleotide polymorphisms and indel variants across a reference BED file, and Nextgene (from Biogene) for comparison with the MiSeq data.

Bottom Line: Predictive biomarker development is a key challenge for novel cancer therapeutics.We found 97% concordance of mutation calls by MiSeq and IT-PGM at a variant allele frequency ⩾13% and ⩾500 × depth coverage, and 91% concordance between MiSeq and Sequenom.Common 'actionable' mutations involved deoxyribonucleic acid (DNA) repair (51%), RAS-RAF-MEK (35%), Wnt (26%), and PI3K-AKT-mTOR (24%) signalling.

View Article: PubMed Central - PubMed

Affiliation: 1] Cancer Biomarkers Team, The Institute of Cancer Research, 15 Cotswold Road, Sutton, Surrey SM2 5NG, UK [2] Drug Development Unit, The Royal Marsden NHS Foundation Trust, Sutton, Surrey SM2 5PT, UK.

ABSTRACT

Background: Predictive biomarker development is a key challenge for novel cancer therapeutics. We explored the feasibility of next-generation sequencing (NGS) to validate exploratory genomic biomarkers that impact phase I trial selection.

Methods: We prospectively enrolled 158 patients with advanced solid tumours referred for phase I clinical trials at the Royal Marsden Hospital (October 2012 to March 2013). After fresh and/or archived tumour tissue were obtained, 93 patients remained candidates for phase I trials. Results from tumour sequencing on the Illumina MiSeq were cross-validated in 27 out of 93 patients on the Ion Torrent Personal Genome Machine (IT-PGM) blinded to results. MiSeq validation with Sequenom MassARRAY OncoCarta 1.0 (Sequenom Inc., San Diego, CA, USA) was performed in a separate cohort.

Results: We found 97% concordance of mutation calls by MiSeq and IT-PGM at a variant allele frequency ⩾13% and ⩾500 × depth coverage, and 91% concordance between MiSeq and Sequenom. Common 'actionable' mutations involved deoxyribonucleic acid (DNA) repair (51%), RAS-RAF-MEK (35%), Wnt (26%), and PI3K-AKT-mTOR (24%) signalling. Out of 53, 29 (55%) patients participating in phase I trials were recommended based on identified actionable mutations.

Conclusions: Targeted high-coverage NGS panels are a highly feasible single-centre technology well-suited to cross-platform validation, enrichment of trials with molecularly defined populations and hypothesis testing early in drug development.

Show MeSH
Related in: MedlinePlus