Limits...
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.

Show MeSH

Related in: MedlinePlus

Effect of blockage and site of block retrieval on quality control parameters for MiSeq. (A) Distribution of DNA-QC scores among samples run on the MiSeq. (B) The effect of blockage on QC scores, showing the best QC scores for fresh tissue obtained in 2013. (C) A plot of QC scores by referring hospital shows that sufficient quality tumour DNA can be extracted from nearly all submitted tumour blocks despite varying laboratory practices. Abbreviations: DNA=deoxyribonucleic acid; QC=quality control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4150267&req=5

fig2: Effect of blockage and site of block retrieval on quality control parameters for MiSeq. (A) Distribution of DNA-QC scores among samples run on the MiSeq. (B) The effect of blockage on QC scores, showing the best QC scores for fresh tissue obtained in 2013. (C) A plot of QC scores by referring hospital shows that sufficient quality tumour DNA can be extracted from nearly all submitted tumour blocks despite varying laboratory practices. Abbreviations: DNA=deoxyribonucleic acid; QC=quality control.

Mentions: Out of 93 patients, 85 (91%) had samples that passed QC for sequencing. Sample QC parameters were not affected adversely by tumour blockage or referral centre (n=58) (Figure 2), although the best QC scores were obtained from fresh tissue sampled in 2013 at site 32 (RMH-DDU). Three hundred and ninety-six mutations were detected from 69 of the 85 samples (81%) that passed QC, while 16 (19%) patients had no mutations detected despite excellent tumour QC parameters (Supplementary Table 4). In total, 232 of these 396 (59%) mutations had been previously described as oncogenic in the COSMIC database; 69 out of 396 (17%) mutations were unreported but impacting an amino acid previously described to be mutated in cancer; 95 out of 396 (24%) were mutations not previously described. Most frequently mutated genes were TP53 (22%), PIK3CA (12%), APC (11%), KRAS (9%), ATM (6%), and FBXW7 (5%) (Figure 3), although a substantial proportion of the mutations seen in ATM and FBXW7 had not been previously described as oncogenic. Mutations in BRAF (3%), MET (3%), KIT (3%), EGFR (3%), ERBB2 (2%), and AKT1 (1%) were also detected.


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)

Effect of blockage and site of block retrieval on quality control parameters for MiSeq. (A) Distribution of DNA-QC scores among samples run on the MiSeq. (B) The effect of blockage on QC scores, showing the best QC scores for fresh tissue obtained in 2013. (C) A plot of QC scores by referring hospital shows that sufficient quality tumour DNA can be extracted from nearly all submitted tumour blocks despite varying laboratory practices. Abbreviations: DNA=deoxyribonucleic acid; QC=quality control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig2: Effect of blockage and site of block retrieval on quality control parameters for MiSeq. (A) Distribution of DNA-QC scores among samples run on the MiSeq. (B) The effect of blockage on QC scores, showing the best QC scores for fresh tissue obtained in 2013. (C) A plot of QC scores by referring hospital shows that sufficient quality tumour DNA can be extracted from nearly all submitted tumour blocks despite varying laboratory practices. Abbreviations: DNA=deoxyribonucleic acid; QC=quality control.
Mentions: Out of 93 patients, 85 (91%) had samples that passed QC for sequencing. Sample QC parameters were not affected adversely by tumour blockage or referral centre (n=58) (Figure 2), although the best QC scores were obtained from fresh tissue sampled in 2013 at site 32 (RMH-DDU). Three hundred and ninety-six mutations were detected from 69 of the 85 samples (81%) that passed QC, while 16 (19%) patients had no mutations detected despite excellent tumour QC parameters (Supplementary Table 4). In total, 232 of these 396 (59%) mutations had been previously described as oncogenic in the COSMIC database; 69 out of 396 (17%) mutations were unreported but impacting an amino acid previously described to be mutated in cancer; 95 out of 396 (24%) were mutations not previously described. Most frequently mutated genes were TP53 (22%), PIK3CA (12%), APC (11%), KRAS (9%), ATM (6%), and FBXW7 (5%) (Figure 3), although a substantial proportion of the mutations seen in ATM and FBXW7 had not been previously described as oncogenic. Mutations in BRAF (3%), MET (3%), KIT (3%), EGFR (3%), ERBB2 (2%), and AKT1 (1%) were also detected.

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