Targeted Next Generation Sequencing as a Reliable Diagnostic Assay for the Detection of Somatic Mutations in Tumours Using Minimal DNA Amounts from Formalin Fixed Paraffin Embedded Material.

Wendy W J De Leng,Edwin Cuppen,Françoise A S Barendregt-Smouter,Maja De Jonge,Ies Nijman,Marlies H G Langenberg,Martijn P Lolkema,Stef Van Lieshout,Stefan M Willems,Robert D Loberg,John W J Hinrichs,Emile E Voest,Neeltje Steeghs,Stefan Sleijfer,Marco J Koudijs,Roel A De Weger,Christa G Gadellaa-Van Hooijdonk,Ajay Goel

doi:10.1371/journal.pone.0149405

Abstract

BackgroundTargeted Next Generation Sequencing (NGS) offers a way to implement testing of multiple genetic aberrations in diagnostic pathology practice, which is necessary for personalized cancer treatment. However, no standards regarding input material have been defined. This study therefore aimed to determine the effect of the type of input material (e.g. formalin fixed paraffin embedded (FFPE) versus fresh frozen (FF) tissue) on NGS derived results. Moreover, this study aimed to explore a standardized analysis pipeline to support consistent clinical decision-making.MethodWe used the Ion Torrent PGM sequencing platform in combination with the Ion AmpliSeq Cancer Hotspot Panel v2 to sequence frequently mutated regions in 50 cancer related genes, and validated the NGS detected variants in 250 FFPE samples using standard diagnostic assays. Next, 386 tumour samples were sequenced to explore the effect of input material on variant detection variables. For variant calling, Ion Torrent analysis software was supplemented with additional variant annotation and filtering.ResultsBoth FFPE and FF tissue could be sequenced reliably with a sensitivity of 99.1%. Validation showed a 98.5% concordance between NGS and conventional sequencing techniques, where NGS provided both the advantage of low input DNA concentration and the detection of low-frequency variants. The reliability of mutation analysis could be further improved with manual inspection of sequence data.ConclusionTargeted NGS can be reliably implemented in cancer diagnostics using both FFPE and FF tissue when using appropriate analysis settings, even with low input DNA.

Highlights

Sequencing the first human genome in 2008 using massive parallel sequencing was suggested to be the first step in personalized medicine.[1]
Targeted Next Generation Sequencing (NGS) can be reliably implemented in cancer diagnostics using both formalin fixed paraffin embedded (FFPE) and fresh frozen (FF) tissue when using appropriate analysis settings, even with low input DNA
Analysis of library statistics showed a significantly increased percentage on-target reads (p = 0.002) for FF samples compared to FFPE samples (Fig 2B), where the number of samples containing a low percentage on-target reads was limited

Summary

Introduction

Sequencing the first human genome in 2008 using massive parallel sequencing was suggested to be the first step in personalized medicine.[1]. Benchtop Generation Sequencing (NGS) platforms and accompanying gene panels are more suitable for routine diagnostics. These platforms provide a cost- and time efficient alternative to classical sequencing techniques like Sanger sequencing.[2, 3] sufficiently powered studies providing evidence that NGS is reliable enough to be used in a diagnostic workflow are lacking. Targeted Generation Sequencing (NGS) offers a way to implement testing of multiple genetic aberrations in diagnostic pathology practice, which is necessary for personalized cancer treatment. This study aimed to determine the effect of the type of input material (e.g. formalin fixed paraffin embedded (FFPE) versus fresh frozen (FF) tissue) on NGS derived results. This study aimed to explore a standardized analysis pipeline to support consistent clinical decision-making

Objectives

Methods

Results

Conclusion