Robust detection of translocations in lymphoma FFPE samples using targeted locus capture-based sequencing

Amin Allahyar ,Roos J Leguit ,Léon C Van Kempen ,Adrien Melquiond ,G Tjitske Los-De Vries ,Erik Splinter ,Phylicia Stathi ,Agata Rakszewska ,Daphne De Jong ,Peter H.l Krijger ,Arjan Diepstra ,Nathalie J Hijmering ,Bauke Ylstra ,Ruud W J Meijers ,Joost S.p Vermaat ,Marieke Simonis ,Paula J P De Vree ,Tom Van Wezel ,Robert Van Der Geize ,Karima Hajo ,Joost F Swennenhuis ,Marjon J.a.m Verstegen ,Mehmet Yılmaz ,Max Van Min ,Mark Pieterse ,Milan Sharma ,Arjen H.g Cleven ,Harma Feitsma ,Wouter De Laat

doi:10.1038/s41467-021-23695-8

Abstract

In routine diagnostic pathology, cancer biopsies are preserved by formalin-fixed, paraffin-embedding (FFPE) procedures for examination of (intra-) cellular morphology. Such procedures inadvertently induce DNA fragmentation, which compromises sequencing-based analyses of chromosomal rearrangements. Yet, rearrangements drive many types of hematolymphoid malignancies and solid tumors, and their manifestation is instructive for diagnosis, prognosis, and treatment. Here, we present FFPE-targeted locus capture (FFPE-TLC) for targeted sequencing of proximity-ligation products formed in FFPE tissue blocks, and PLIER, a computational framework that allows automated identification and characterization of rearrangements involving selected, clinically relevant, loci. FFPE-TLC, blindly applied to 149 lymphoma and control FFPE samples, identifies the known and previously uncharacterized rearrangement partners. It outperforms fluorescence in situ hybridization (FISH) in sensitivity and specificity, and shows clear advantages over standard capture-NGS methods, finding rearrangements involving repetitive sequences which they typically miss. FFPE-TLC is therefore a powerful clinical diagnostics tool for accurate targeted rearrangement detection in FFPE specimens.

Highlights

In routine diagnostic pathology, cancer biopsies are preserved by formalin-fixed, paraffinembedding (FFPE) procedures for examination of cellular morphology
As an assay to be applied in the diagnostic setting, FFPE-TLC offers important advantages over fluorescence in situ hybridization (FISH), the current gold standard for targeted rearrangement detection in lymphoma FFPE samples
Unlike FFPE-TLC, FISH is highly dependent on good quality tissue and cell morphology, which may be negatively impacted by necrosis, apoptosis, and crush artifacts in resection specimens and by very limited material from core needle biopsy samples

Summary

Introduction

Cancer biopsies are preserved by formalin-fixed, paraffinembedding (FFPE) procedures for examination of (intra-) cellular morphology. FFPE-TLC, blindly applied to 149 lymphoma and control FFPE samples, identifies the known and previously uncharacterized rearrangement partners It outperforms fluorescence in situ hybridization (FISH) in sensitivity and specificity, and shows clear advantages over standard capture-NGS methods, finding rearrangements involving repetitive sequences which they typically miss. Next-generation sequencing (NGS) DNA capture methods have been introduced for rearrangement detection in selected gene panels in FFPE samples, which makes it possible to detect breakpoints at base-pair resolution and identify translocation partner genes[7,8,9,10]. Proof-of-concept that proximity-ligation methods can detect SVs in FFPE material was recently provided in a non-blind study that applied a Hi-C protocol (i.e., a genome-wide variant of proximity-ligation assays) to 15 FFPE tumor samples In most cases, this method (called “Fix-C”) gave visually appreciable altered contact frequencies in genes previously scored to harbor rearrangement by FISH24. FFPETLC is a powerful tool for SV detection in FFPE samples in malignant lymphoma and other translocation-mediated malignancies

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Conclusion