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Review
. 2022 May 5;12(5):750.
doi: 10.3390/jpm12050750.

FFPE-Based NGS Approaches into Clinical Practice: The Limits of Glory from a Pathologist Viewpoint

Filippo Cappello  1 , Valentina Angerilli  1 , Giada Munari  2 , Carlotta Ceccon  1 , Marianna Sabbadin  2 , Fabio Pagni  3 , Nicola Fusco  4   5 , Umberto Malapelle  6 , Matteo Fassan  1   2
Affiliations
Review

FFPE-Based NGS Approaches into Clinical Practice: The Limits of Glory from a Pathologist Viewpoint

Filippo Cappello et al. J Pers Med. .

Abstract

The introduction of next-generation sequencing (NGS) in the molecular diagnostic armamentarium is deeply changing pathology practice and laboratory frameworks. NGS allows for the comprehensive molecular characterization of neoplasms, in order to provide the best treatment to oncologic patients. On the other hand, NGS raises technical issues and poses several challenges in terms of education, infrastructures and costs. The aim of this review is to give an overview of the main NGS sequencing platforms that can be used in current molecular diagnostics and gain insights into the clinical applications of NGS in precision oncology. Hence, we also focus on the preanalytical, analytical and interpretative issues raised by the incorporation of NGS in routine pathology diagnostics.

Keywords: NGS; biomarkers; diagnostics; precision medicine.

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Conflict of interest statement

Umberto Malapelle received personal fees (as a consultant and/or speaker bureau) from Boehringer Ingelheim, Roche, MSD, Amgen, Thermo Fisher Scientifics, Eli Lilly, Diaceutics, GSK, Merck, AstraZeneca and Jannseen (unrelated to the current work). Nicola Fusco received honoraria for their consulting, advisory role and/or speaker bureau from Merck Sharp & Dohme (MSD), Boehringer Ingelheim, Novartis, AstraZeneca, and Daiichi Sankyo (unrelated to the current work). Fabio Pagni received honoraria for their consulting, advisory role and/or speaker bureau from Merck Sharp & Dohme (MSD), Novartis, Roche, and Amgen (unrelated to the current work). Matteo Fassan received research funding from Astellas Pharma, Macrophage Pharma and QED Therapeutics and had roles as a consultant or advisor for Astellas Pharma, Roche, Astra Zeneca, MSD and GSK-Tesaro (unrelated to the current work). All the other authors declare no conflict of interest regarding the publication of this article.

Figures

Figure 1
Figure 1
Representative images of potential pitfalls in NGS analysis of FFPE bioptic and surgical specimens. (A,B) Hematic material enclosing few adenocarcinoma glands; (C) biopsy specimen composed of fibrotic tissue enclosing rare adenocarcinoma glands; (D) scattered tumor cells surrounded by necrosis and fibrosis in a metastatic surgical resection specimen; (E) mucinous adenocarcinoma characterized by low cellularity and mucinous acellular component; (F) intratumor heterogeneity may hamper NGS analysis if both components are not considered (a morphologically heterogeneous colorectal adenocarcinoma composed by glandular and solid areas is shown).
Figure 2
Figure 2
Representative images of FFPE tissue blocks inadequate for NGS analysis due to (A) scarcity of material (B) and absence of material following previous sectioning for diagnostic purposes. (C) A hub center for NGS-based molecular diagnostics receives different types of FFPE tissue specimens (i.e., biopsy, surgical resection and cytology specimens) obtained with different workflows and processes.
Figure 3
Figure 3
An exemplificative case of EGFR exon 19 deletion p.A746_A750 del detected by using NGS Ion Torrent S5 (Thermo Fisher Scientifics) platform. In this figure, loading density (A), technical quality parameters (B), read length histogram (C) and visual inspection of detected mutations with Golden Helix Genome Browse tool (D) were observed.
See this image and copyright information in PMC

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