Dataset Information

The Mutational Concordance of Fixed Formalin Paraffin Embedded and Fresh Frozen Gastro-Oesophageal Tumours Using Whole Exome Sequencing.

ABSTRACT: 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 (N = 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 (p < 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/?-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.

SUBMITTER: Chong IY

PROVIDER: S-EPMC7826535 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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Publications

The Mutational Concordance of Fixed Formalin Paraffin Embedded and Fresh Frozen Gastro-Oesophageal Tumours Using Whole Exome Sequencing.

Chong Irene Y IY Starling Naureen N Rust Alistair A Alexander John J Aronson Lauren L Llorca-Cardenosa Marta M Chauhan Ritika R Chaudry Asif A Kumar Sacheen S Fenwick Kerry K Assiotis Ioannis I Matthews Nik N Begum Ruwaida R Wotherspoon Andrew A Terlizzo Monica M Watkins David D Chau Ian I Lord Christopher J CJ Haider Syed S Rao Sheela S Cunningham David D

Journal of clinical medicine 20210109 2

1.<h4>Background</h4>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 sa ...[more]

PMID: 33435284

Similar Datasets

Project description:BACKGROUND: Gene expression profiling of formalin-fixed, paraffin-embedded (FFPE) samples represents a valuable approach for advancing oncology diagnostics and enhancing retrospective clinical studies; however, at present, this methodology still requires optimization and thus has not been extensively used. Here, we utilized thorough quality control methods to assess RNA extracted from FFPE samples and then compared it to RNA extracted from matched fresh-frozen (FF) counterparts. We preformed genome-wide expression profiling of FF and FFPE ovarian serous adenocarcinoma sample pairs and compared their gene signatures to normal ovary samples. METHODS: RNA from FFPE samples was extracted using two different methods, Ambion and Agencourt, and its quality was determined by profiling starting total RNA on Bioanalyzer and by amplifying increasing size fragments of beta actin (ACTB) and claudin 3 (CLDN3) by reverse-transcriptase polymerase chain reaction. Five matched FF and FFPE ovarian serous adenocarcinoma samples, as well as a set of normal ovary samples, were profiled using whole genome Agilent microarrays. Reproducibility of the FF and FFPE replicates was measured using Pearson correlation, whereas comparison between the FF and FFPE samples was done using a Z-score analysis. RESULTS: Data analysis showed high reproducibility of expression within each FF and FFPE method, whereas matched FF and FFPE pairs demonstrated lower similarity, emphasizing an inherent difference between the two sample types. Z-score analysis of matched FF and FFPE samples revealed good concordance of top 100 differentially expressed genes with the highest correlation of 0.84. Genes characteristic of ovarian serous adenocarcinoma, including a well known marker CLDN3, as well as potentially some novel markers, were identified by comparing gene expression profiles of ovarian adenocarcinoma to those of normal ovary. CONCLUSION: Conclusively, we showed that systematic assessment of FFPE samples at the RNA level is essential for obtaining good quality gene expression microarray data. We also demonstrated that profiling of not only FF but also of FFPE samples can be successfully used to identify differentially expressed genes characteristic of ovarian carcinoma.

Dataset Information

The Mutational Concordance of Fixed Formalin Paraffin Embedded and Fresh Frozen Gastro-Oesophageal Tumours Using Whole Exome Sequencing.

Background

Methods

Results

Conclusions

Publications

The Mutational Concordance of Fixed Formalin Paraffin Embedded and Fresh Frozen Gastro-Oesophageal Tumours Using Whole Exome Sequencing.

Similar Datasets

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