The Mutational Concordance of Fixed Formalin Paraffin Embedded and Fresh Frozen Gastro-Oesophageal Tumours Using Whole Exome Sequencing
1. Background: The application of massively parallel sequencing has led to the identification of aberrant druggable pathways and somatic mutations within therapeutically relevant genes in gastro-oesophageal cancer. Given the widespread use of formalin-fixed paraffin-embedded (FFPE) samples in the st...
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Language: | English |
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MDPI AG
2021-01-01
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Series: | Journal of Clinical Medicine |
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Online Access: | https://www.mdpi.com/2077-0383/10/2/215 |
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doaj-915bb413902f4856b83979729b3dfa2c |
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record_format |
Article |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Irene Y. Chong Naureen Starling Alistair Rust John Alexander Lauren Aronson Marta Llorca-Cardenosa Ritika Chauhan Asif Chaudry Sacheen Kumar Kerry Fenwick Ioannis Assiotis Nik Matthews Ruwaida Begum Andrew Wotherspoon Monica Terlizzo David Watkins Ian Chau Christopher J. Lord Syed Haider Sheela Rao David Cunningham |
spellingShingle |
Irene Y. Chong Naureen Starling Alistair Rust John Alexander Lauren Aronson Marta Llorca-Cardenosa Ritika Chauhan Asif Chaudry Sacheen Kumar Kerry Fenwick Ioannis Assiotis Nik Matthews Ruwaida Begum Andrew Wotherspoon Monica Terlizzo David Watkins Ian Chau Christopher J. Lord Syed Haider Sheela Rao David Cunningham The Mutational Concordance of Fixed Formalin Paraffin Embedded and Fresh Frozen Gastro-Oesophageal Tumours Using Whole Exome Sequencing Journal of Clinical Medicine gastro-oesophageal cancer mutational concordance exome sequencing formalin fixed paraffin embedded biomarkers |
author_facet |
Irene Y. Chong Naureen Starling Alistair Rust John Alexander Lauren Aronson Marta Llorca-Cardenosa Ritika Chauhan Asif Chaudry Sacheen Kumar Kerry Fenwick Ioannis Assiotis Nik Matthews Ruwaida Begum Andrew Wotherspoon Monica Terlizzo David Watkins Ian Chau Christopher J. Lord Syed Haider Sheela Rao David Cunningham |
author_sort |
Irene Y. Chong |
title |
The Mutational Concordance of Fixed Formalin Paraffin Embedded and Fresh Frozen Gastro-Oesophageal Tumours Using Whole Exome Sequencing |
title_short |
The Mutational Concordance of Fixed Formalin Paraffin Embedded and Fresh Frozen Gastro-Oesophageal Tumours Using Whole Exome Sequencing |
title_full |
The Mutational Concordance of Fixed Formalin Paraffin Embedded and Fresh Frozen Gastro-Oesophageal Tumours Using Whole Exome Sequencing |
title_fullStr |
The Mutational Concordance of Fixed Formalin Paraffin Embedded and Fresh Frozen Gastro-Oesophageal Tumours Using Whole Exome Sequencing |
title_full_unstemmed |
The Mutational Concordance of Fixed Formalin Paraffin Embedded and Fresh Frozen Gastro-Oesophageal Tumours Using Whole Exome Sequencing |
title_sort |
mutational concordance of fixed formalin paraffin embedded and fresh frozen gastro-oesophageal tumours using whole exome sequencing |
publisher |
MDPI AG |
series |
Journal of Clinical Medicine |
issn |
2077-0383 |
publishDate |
2021-01-01 |
description |
1. Background: The application of massively parallel sequencing has led to the identification of aberrant druggable pathways and somatic mutations within therapeutically relevant genes in gastro-oesophageal cancer. Given the widespread use of formalin-fixed paraffin-embedded (FFPE) samples in the study of this disease, it would be beneficial, especially for the purposes of biomarker evaluation, to assess the concordance between comprehensive exome-wide sequencing data from archival FFPE samples originating from a prospective clinical study and those derived from fresh-frozen material. 2. Methods: We analysed whole-exome sequencing data to define the mutational concordance of 16 matched fresh-frozen and FFPE gastro-oesophageal tumours (<i>N</i> = 32) from a prospective clinical study. We assessed DNA integrity prior to sequencing and then identified coding mutations in genes that have previously been implicated in other cancers. In addition, we calculated the mutant-allele heterogeneity (MATH) for these samples. 3. Results: Although there was increased degradation of DNA in FFPE samples compared with frozen samples, sequencing data from only two FFPE samples failed to reach an adequate mapping quality threshold. Using a filtering threshold of mutant read counts of at least ten and a minimum of 5% variant allele frequency (VAF) we found that there was a high median mutational concordance of 97% (range 80.1–98.68%) between fresh-frozen and FFPE gastro-oesophageal tumour-derived exomes. However, the majority of FFPE tumours had higher mutant-allele heterogeneity (MATH) scores when compared with corresponding frozen tumours (<i>p</i> < 0.001), suggesting that FFPE-based exome sequencing is likely to over-represent tumour heterogeneity in FFPE samples compared to fresh-frozen samples. Furthermore, we identified coding mutations in 120 cancer-related genes, including those associated with chromatin remodelling and Wnt/β-catenin and Receptor Tyrosine Kinase signalling. 4. Conclusions: These data suggest that comprehensive genomic data can be generated from exome sequencing of selected DNA samples extracted from archival FFPE gastro-oesophageal tumour tissues within the context of prospective clinical trials. |
topic |
gastro-oesophageal cancer mutational concordance exome sequencing formalin fixed paraffin embedded biomarkers |
url |
https://www.mdpi.com/2077-0383/10/2/215 |
work_keys_str_mv |
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doaj-915bb413902f4856b83979729b3dfa2c2021-01-10T00:00:21ZengMDPI AGJournal of Clinical Medicine2077-03832021-01-011021521510.3390/jcm10020215The Mutational Concordance of Fixed Formalin Paraffin Embedded and Fresh Frozen Gastro-Oesophageal Tumours Using Whole Exome SequencingIrene Y. Chong0Naureen Starling1Alistair Rust2John Alexander3Lauren Aronson4Marta Llorca-Cardenosa5Ritika Chauhan6Asif Chaudry7Sacheen Kumar8Kerry Fenwick9Ioannis Assiotis10Nik Matthews11Ruwaida Begum12Andrew Wotherspoon13Monica Terlizzo14David Watkins15Ian Chau16Christopher J. Lord17Syed Haider18Sheela Rao19David Cunningham20The Division of Molecular Pathology, The Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, UKThe Royal Marsden Hospital NHS Foundation Trust, 203 Fulham Road, London SW3 6JJ, UKThe Tissue Profiling Unit, The Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, UKBreast Cancer Now Toby Robins Research Centre, The Institute of Cancer Research, London SW3 6JB, UKThe Division of Molecular Pathology, The Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, UKThe Division of Molecular Pathology, The Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, UKThe Tissue Profiling Unit, The Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, UKThe Royal Marsden Hospital NHS Foundation Trust, 203 Fulham Road, London SW3 6JJ, UKThe Royal Marsden Hospital NHS Foundation Trust, 203 Fulham Road, London SW3 6JJ, UKThe Tissue Profiling Unit, The Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, UKThe Tissue Profiling Unit, The Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, UKThe Tissue Profiling Unit, The Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, UKThe Royal Marsden Hospital NHS Foundation Trust, 203 Fulham Road, London SW3 6JJ, UKThe Royal Marsden Hospital NHS Foundation Trust, 203 Fulham Road, London SW3 6JJ, UKThe Royal Marsden Hospital NHS Foundation Trust, 203 Fulham Road, London SW3 6JJ, UKThe Royal Marsden Hospital NHS Foundation Trust, 203 Fulham Road, London SW3 6JJ, UKThe Royal Marsden Hospital NHS Foundation Trust, 203 Fulham Road, London SW3 6JJ, UKBreast Cancer Now Toby Robins Research Centre, The Institute of Cancer Research, London SW3 6JB, UKBreast Cancer Now Toby Robins Research Centre, The Institute of Cancer Research, London SW3 6JB, UKThe Royal Marsden Hospital NHS Foundation Trust, 203 Fulham Road, London SW3 6JJ, UKThe Royal Marsden Hospital NHS Foundation Trust, 203 Fulham Road, London SW3 6JJ, UK1. Background: The application of massively parallel sequencing has led to the identification of aberrant druggable pathways and somatic mutations within therapeutically relevant genes in gastro-oesophageal cancer. Given the widespread use of formalin-fixed paraffin-embedded (FFPE) samples in the study of this disease, it would be beneficial, especially for the purposes of biomarker evaluation, to assess the concordance between comprehensive exome-wide sequencing data from archival FFPE samples originating from a prospective clinical study and those derived from fresh-frozen material. 2. Methods: We analysed whole-exome sequencing data to define the mutational concordance of 16 matched fresh-frozen and FFPE gastro-oesophageal tumours (<i>N</i> = 32) from a prospective clinical study. We assessed DNA integrity prior to sequencing and then identified coding mutations in genes that have previously been implicated in other cancers. In addition, we calculated the mutant-allele heterogeneity (MATH) for these samples. 3. Results: Although there was increased degradation of DNA in FFPE samples compared with frozen samples, sequencing data from only two FFPE samples failed to reach an adequate mapping quality threshold. Using a filtering threshold of mutant read counts of at least ten and a minimum of 5% variant allele frequency (VAF) we found that there was a high median mutational concordance of 97% (range 80.1–98.68%) between fresh-frozen and FFPE gastro-oesophageal tumour-derived exomes. However, the majority of FFPE tumours had higher mutant-allele heterogeneity (MATH) scores when compared with corresponding frozen tumours (<i>p</i> < 0.001), suggesting that FFPE-based exome sequencing is likely to over-represent tumour heterogeneity in FFPE samples compared to fresh-frozen samples. Furthermore, we identified coding mutations in 120 cancer-related genes, including those associated with chromatin remodelling and Wnt/β-catenin and Receptor Tyrosine Kinase signalling. 4. Conclusions: These data suggest that comprehensive genomic data can be generated from exome sequencing of selected DNA samples extracted from archival FFPE gastro-oesophageal tumour tissues within the context of prospective clinical trials.https://www.mdpi.com/2077-0383/10/2/215gastro-oesophageal cancermutational concordanceexome sequencingformalin fixed paraffin embeddedbiomarkers |