Formalin-Fixed and Paraffin-Embedded Samples for Next Generation Sequencing: Problems and Solutions.

Gerardo Cazzato,Antonietta Cimmino,Leonardo Resta,Anna Colagrande,Francesca Arezzo,Giuseppe Ingravallo,Paolo Romita,Gennaro Cormio,Paola Parente,Eugenio Maiorano,Maricla Marrone,Concetta Caporusso,Aurora De Marco,Alessandra Stellacci,Roberta Rossi,Vincenzo Venerito,Teresa Lettini,Caterina Foti,Vera Loizzi,Vincenza Sara Scarcella ,Piero Tarantino

doi:10.3390/genes12101472

Abstract

Over the years, increasing information has been asked of the pathologist: we have moved from a purely morphological diagnosis to biomolecular and genetic studies, which have made it possible to implement the use of molecular targeted therapies, such as anti-epidermal growth factor receptor (EGFR) molecules in EGFR-mutated lung cancer, for example. Today, next generation sequencing (NGS) has changed the approach to neoplasms, to the extent that, in a short time, it has gained a place of absolute importance and diagnostic, prognostic and therapeutic utility. In this scenario, formaldehyde-fixed and paraffin-embedded (FFPE) biological tissue samples are a source of clinical and molecular information. However, problems can arise in the genetic material (DNA and RNA) for use in NGS due to fixation, and work is being devoted to possible strategies to reduce its effects. In this paper, we discuss the applications of FFPE tissue samples in the execution of NGS, we focus on the problems arising with the use of this type of material for nucleic acid extraction and, finally, we consider the most useful strategies to prevent and reduce single nucleotide polymorphisms (SNV) and other fixation artifacts.

Highlights

In recent decades, a complete change in diagnostic techniques has occurred in various sectors of medicine
We discuss the applications of formaldehyde-fixed and paraffin-embedded (FFPE) tissue samples in the execution of next generation sequencing (NGS), we focus on the problems arising with the use of this type of material for nucleic acid extraction and, we consider the most useful strategies to prevent and reduce single nucleotide polymorphisms (SNV) and other fixation artifacts
This study demonstrated the feasibility of calling and filtering genetic variants from FFPE tissue samples using a combined strategy with molecular barcodes, optimized DNA extraction, and bioinformatics methods that incorporate the genomic context such as the mutational signature and variant allelic frequency [32,37]

Summary

Introduction

A complete change in diagnostic techniques has occurred in various sectors of medicine. Generation sequencing (NGS) [1] is certainly gaining an increasingly important role, to the extent that we have moved on from only morphological characterization of tumors, this is still fundamental, to a more detailed and extensive analysis of a constantly increasing number of genes [2]. As technical aspects are being perfected, this technique is becoming ever faster and more efficient [4]. In this perspective, tissues fixed in formaldehyde and included in paraffin (FFPE) have a role of absolute importance. We focus on these aspects, taking as reference the most recent discoveries in the scientific field, and discuss the limitations regarding the use of FFPE for NGS that still exist, while taking a look at possible scenarios in the near and distant future

Methods

Results

Conclusion