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Formalin-Fixed Paraffin-Embedded Tissue (FFPET) Sections for Nucleic Acid-Based Analysis in Biomarker Discovery and Early Drug Development

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General Methods in Biomarker Research and their Applications

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Abstract

Formalin-fixed paraffin-embedded tissue (FFPET) samples represent the clinical standard of tissue fixation, and huge archives of this material offer a valuable source for biomarker identification and validation. For this reason, techniques for efficient and reliable analysis of FFPET are important for rapid advances in personalized health care (PHC). This chapter focuses on the use of FFPET for nucleic acid biomarker verification by RNA and DNA analysis in clinical FFPET samples, describes in particular the workflow applied for hypothesis testing to determine quantitative expression levels of putative transcriptional biomarkers in different tumor FFPET samples, and gives an overview of current major applications for nucleic acid analyses from FFPET. Due to formalin fixation-caused modification and degradation of RNA and DNA, special attention is paid to the methods for nucleic acid isolation, because yield and quality of extracted nucleic acids are crucial for successful downstream applications. The described procedures usually achieve yields of several μg of high-quality RNA and DNA obtained from standard 5–10 μm FFPET sections; however, amounts significantly depend on factors like sample size, number of well-preserved cells, and tumor entity. A major application for isolated RNA is its use for reliable quantitative gene expression analysis by reverse transcription quantitative real-time PCR (RT-qPCR) assays based on optimized cDNA synthesis and quantitative real-time PCR (qPCR). In cases of very low starting material, an optimized pre-amplification protocol achieves an up to 4,000-fold increase in cDNA yields, thereby significantly improving the sensitivity of downstream applications. Pre-amplification is also implemented in a workflow for RT-qPCR analysis of microdissected material from immunohistochemically stained FFPET sections. This procedure permits capture of defined cell types in order to enhance specificity of gene expression profiling. Further applications for RNA analysis relate to expression profiling by microarrays and to a combination of genetic and transcriptional analysis by advanced RNA-Seq technology utilizing next-generation sequencing (NGS). The major applications described for DNA isolated from FFPET relate to qPCR and various approaches for mutation analysis. The overview comprises sequencing techniques including NGS as well as high-resolution melting (HRM) and allele-specific qPCR (AS qPCR). Additionally the use of FFPET for the analysis of epigenetic methylation patterns by methylation-sensitive HRM (MS-HRM) is addressed. The final chapter refers to the application of FFPET in clinical diagnostics of different cancer types. To date first approved IVD kits are available to analyze mutations as well as expression patterns of related marker genes and thus represent an essential precondition for prognosis, treatment decisions, and therapy in PHC.

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Abbreviations

AS qPCR:

Allele-Specific qPCR

CGH:

Comparative Genomic Hybridization

Cq:

Quantification Cycle

CRC:

Colorectal Cancer

FFPET:

Formalin-Fixed Paraffin-Embedded Tissue

HPRT1:

Hypoxanthine Phosphoribosyltransferase 1

HRM:

High-Resolution Melting

LCM:

Laser Capture Microdissection

MRPL19:

Mitochondrial Ribosomal Protein L19

MS-HRM:

Methylation-Sensitive High-Resolution Melting

NGS:

Next Generation Sequencing

NSCLC:

Non-small Cell Lung Cancer

PHC:

Personalized Health Care

qPCR:

Quantitative Real-Time PCR

RIN:

RNA Integrity Number

RT-qPCR:

Reverse Transcription qPCR

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Authors and Affiliations

  1. Roche Diagnostics GmbH, Werk Penzberg, Nonnenwald 2, D-82377, Penzberg, Germany

    Sabine Lohmann, Beatrix Bahle, Andrea Herold & Julian Schuster

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  1. Sabine Lohmann
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  2. Beatrix Bahle
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  3. Andrea Herold
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  4. Julian Schuster
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Correspondence to Sabine Lohmann .

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Editors and Affiliations

  1. Department of Nutrition and Dietetics, Division of Diabetes & Nutritional Sciences, Faculty of Life Sciences & Medicine, King's College London, London, United Kingdom

    Victor R. Preedy

  2. Faculty of Science & Technology, Department of Biomedical Sciences, University of Westminster, London, United Kingdom

    Vinood B. Patel

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Lohmann, S., Bahle, B., Herold, A., Schuster, J. (2015). Formalin-Fixed Paraffin-Embedded Tissue (FFPET) Sections for Nucleic Acid-Based Analysis in Biomarker Discovery and Early Drug Development. In: Preedy, V., Patel, V. (eds) General Methods in Biomarker Research and their Applications. Biomarkers in Disease: Methods, Discoveries and Applications. Springer, Dordrecht. https://doi.org/10.1007/978-94-007-7696-8_24

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