Assessment of the quality of DNA from various formalin-fixed paraffin-embedded (FFPE) tissues and the use of this DNA for next-generation sequencing (NGS) with no artifactual mutation

Einaga, Naoki; Yoshïda, Akio; Noda, Hiroko; Suemitsu, Maki; Nakayama, Yuki; Sakurada, Akira; Kawaji, Yoshiko; Yamaguchi, Hiromi; Sasaki, Yasushi; Tokino, Takashi; Esumi, Mariko

doi:10.1371/journal.pone.0176280

Cited by 90 publications

(78 citation statements)

References 26 publications

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“…We utilised the TSO500 assay in order to understand its utility and accuracy in determining both the TMB and druggable mutation calls in cancer. One of the key challenges with patient testing [23] is the ability to take a patient biopsy sample with limited input material and produce sequencing data and mutational calls of sufficient quality in order to make decisions on target selection and drug therapy [24].…”

Section: Discussionmentioning

confidence: 99%

Use of an Integrated Pan-Cancer Oncology Enrichment Next-Generation Sequencing Assay to Measure Tumour Mutational Burden and Detect Clinically Actionable Variants

et al. 2020

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Introduction The identification of tumour mutational burden (TMB) as a biomarker of response to programmed cell death protein 1 (PD-1) immunotherapy has necessitated the development of genomic assays to measure this. We carried out comprehensive molecular profiling of cancers using the Illumina TruSight Oncology 500 (TSO500) panel and compared these to whole-genome sequencing (WGS). Methods Cancer samples derived from formalin-fixed material were profiled on the TSO500 panel, sequenced on an Illumina NextSeq 500 instrument and processed through the TSO500 Docker pipeline. Either FASTQ files (PierianDx) or vcf files (OncoKDM) were processed to understand clinical actionability. Results In total, 108 samples (a mixture of colorectal, lung, oesophageal and control samples) were processed via the DNA panel. There was good correlation between TMB, single-nucleotide variants (SNVs), indels and copy-number variations as predicted by TSO500 and WGS (R 2 > 0.9) and good reproducibility, with less than 5% variability between repeated controls. For the RNA panel, 13 samples were processed, with all known fusions observed via orthogonal techniques. For clinical actionability, 72 tier 1 variants and 297 tier 2 variants were detected, with clinical trials identified for all patients. Conclusions The TSO500 assay accurately measures TMB, microsatellite instability, SNVs, indels, copy-number/structural variation and gene fusions when compared to WGS and orthogonal technologies. Coupled with a clinical annotation pipeline, this provides a powerful methodology for identification of clinically actionable variants.

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Section: Discussionmentioning

confidence: 99%

Use of an Integrated Pan-Cancer Oncology Enrichment Next-Generation Sequencing Assay to Measure Tumour Mutational Burden and Detect Clinically Actionable Variants

et al. 2020

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“…For example, low tumor purity, which can result from infiltration of immune or tumor microenvironment cells, can lead to reduced TMB assay sensitivity. Also, fixation time is a preanalytical factor that influences the introduction of FFPE‐induced deamination artefacts, which also impacts TMB estimation at the stage of bioinformatic analysis …”

Section: Variation In Tmb Assessment and Factors That Impact Tmb Outputmentioning

confidence: 99%

“…Also, fixation time is a preanalytical factor that influences the introduction of FFPE-induced deamination artefacts, which also impacts TMB estimation at the stage of bioinformatic analysis. 58,59 For sequencing, genome coverage differs between WGS, WES, and targeted gene panel assays. WGS covers the whole genome, WES covers the entire exome coding region, and targeted gene panels cover specified areas that may or may not include tumor suppressor genes, driver genes, or intronic regions.…”

Section: Variation In Tmb Assessment and Factors That Impact Tmb Oumentioning

confidence: 99%

Tumor mutational burden standardization initiatives: Recommendations for consistent tumor mutational burden assessment in clinical samples to guide immunotherapy treatment decisions

Stenzinger

Allen

Maas³

et al. 2019

Genes Chromosomes & Cancer

188

161

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Characterization of tumors utilizing next‐generation sequencing methods, including assessment of the number of somatic mutations (tumor mutational burden [TMB]), is currently at the forefront of the field of personalized medicine. Recent clinical studies have associated high TMB with improved patient response rates and survival benefit from immune checkpoint inhibitors; hence, TMB is emerging as a biomarker of response for these immunotherapy agents. However, variability in current methods for TMB estimation and reporting is evident, demonstrating a need for standardization and harmonization of TMB assessment methodology across assays and centers. Two uniquely placed organizations, Friends of Cancer Research (Friends) and the Quality Assurance Initiative Pathology (QuIP), have collaborated to coordinate efforts for international multistakeholder initiatives to address this need. Friends and QuIP, who have partnered with several academic centers, pharmaceutical organizations, and diagnostic companies, have adopted complementary, multidisciplinary approaches toward the goal of proposing evidence‐based recommendations for achieving consistent TMB estimation and reporting in clinical samples across assays and centers. Many factors influence TMB assessment, including preanalytical factors, choice of assay, and methods of reporting. Preliminary analyses highlight the importance of targeted gene panel size and composition, and bioinformatic parameters for reliable TMB estimation. Herein, Friends and QuIP propose recommendations toward consistent TMB estimation and reporting methods in clinical samples across assays and centers. These recommendations should be followed to minimize variability in TMB estimation and reporting, which will ensure reliable and reproducible identification of patients who are likely to benefit from immune checkpoint inhibitors.

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“…Furthermore, we observed that overnight digestion at 65°C improved the efficiency of DNA extraction from FFPE tissues. 12 Phenol chloroform extraction is the conventional method for organic extraction of nucleic acids. However, we found better extraction efficacy with phenol:chloroform: isoamyl alcohol rather than with phenol chloroform extraction (unpublished results).…”

Section: Discussionmentioning

confidence: 99%

PCR Amplifiable DNA from Breast Disease FFPE Section for Mutational Analysis

Panchal

Bhale

Chowdary³

et al. 2020

J Biomol Tech

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Formalin-fixed paraffin-embedded (FFPE) tissue specimens have been a staple of research, providing precious resources for molecular and genomic studies. However, the biggest challenge is the extraction of high-quality DNA from FFPE tissues, given that the integrity of DNA is critically affected by formalin fixation. Formaldehyde induces crosslinks in DNA that renders single or double-stranded DNA breaks. Such breaks cause extensive fragmentation that directly influences the quality of DNA purified and the number of templates available for PCR amplification. Thus, protocol for DNA purification from FFPE tissues must effectively extract highly fragmented DNA and reverse cross-linking caused by formalin fixation. DNA extraction methods available in the literature were selected and modified at different stages to optimize a protocol that extracts DNA of sufficient quality and fragment size to be detectable by PCR. Archived FFPE tissues belonged to patients with triple negative breast cancer (TNBC) and benign breast disease were used for the protocol optimization. The best optimized protocol was then used to amplify Exon 4 region of Proviral integration site for Moloney murine leukemia virus1 (Pim1) kinase gene to analyze any probable somatic mutations both in TNBCs and benign breast diseases. Of the 12 different protocols developed, best quality DNA in terms of fragment size and purity was obtained when Tween20 lysis buffer was used for both deparaffinization and overnight digestion along with high salt precipitation. Optimized protocol was then validated by extracting DNAs from 10 TNBCs and 5 benign breast disease specimens with consistent purity and fragment size. PCR amplification and subsequent Sanger's sequencing revealed the presence of mutations in the Exon 4 region of Pim1 kinase. Deparaffinization and overnight digestion in Tween20 lysis buffer along with high salt precipitation yielded the best quality PCR amplifiable DNA for mutational analysis.

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Assessment of the quality of DNA from various formalin-fixed paraffin-embedded (FFPE) tissues and the use of this DNA for next-generation sequencing (NGS) with no artifactual mutation

Cited by 90 publications

References 26 publications

Use of an Integrated Pan-Cancer Oncology Enrichment Next-Generation Sequencing Assay to Measure Tumour Mutational Burden and Detect Clinically Actionable Variants

Use of an Integrated Pan-Cancer Oncology Enrichment Next-Generation Sequencing Assay to Measure Tumour Mutational Burden and Detect Clinically Actionable Variants

Tumor mutational burden standardization initiatives: Recommendations for consistent tumor mutational burden assessment in clinical samples to guide immunotherapy treatment decisions

PCR Amplifiable DNA from Breast Disease FFPE Section for Mutational Analysis

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