Epigenomic profiling of archived FFPE tissues by enhanced PAT-ChIP (EPAT-ChIP) technology

Stefano Amatori,Claudio Paolicelli,Nora Sahnane,Saverio Minucci,Giuseppe Persico,Roman Hillje,Marco Giorgio,Daniela Furlan,Francesco Corini,Mirco Fanelli,Lucilla Luzi,Pier Giuseppe Pelicci

doi:10.1186/s13148-018-0576-y

Abstract

BackgroundThe introduction of pathology tissue-chromatin immunoprecipitation (PAT-ChIP), a technique allowing chromatin immunoprecipitation (ChIP) from formalin-fixed paraffin-embedded (FFPE) tissues, has extended the application of chromatin studies to clinical patient samples. However, extensive crosslinking introduced during routine tissue fixation of clinical specimens may hamper the application of PAT-ChIP to genome-wide studies (PAT-ChIP-Seq) from archived tissue samples. The reduced efficiency in chromatin extraction from over-fixed formalin archival samples is the main hurdle to overcome, especially when low abundant epigenetic marks (e.g., H3K4me3) are investigated.ResultsWe evaluated different modifications of the original PAT-ChIP protocol to improve chromatin isolation from FFPE tissues. With this aim, we first made extensive usage of a normal human colon specimen fixed at controlled conditions (24 h, 48 h, and 72 h) to mimic the variability of tissue fixation that is most frequently found in archived samples. Different conditions of chromatin extraction were tested applying either diverse sonication protocols or heat-mediated limited reversal of crosslinking (LRC). We found that, if compared with canonical PAT-ChIP protocol, LRC strongly increases chromatin extraction efficiency, especially when 72-h fixed FFPE samples are used. The new procedure, that we named enhanced PAT-ChIP (EPAT-ChIP), was then applied at genome-wide level using an archival sample of invasive breast carcinoma to investigate H3K4me3, a lowly abundant histone modification, and H3K27me3 and H3K27ac, two additional well-known histone marks.ConclusionsEPAT-ChIP procedure improves the efficiency of chromatin isolation from FFPE samples allowing the study of long time-fixed specimens (72 h), as well as the investigation of low distributed epigenetic marks (e.g., H3K4me3) and the analysis of multiple histone marks from low amounts of starting material. We believe that EPAT-ChIP will facilitate the application of chromatin studies to archived pathology samples, thus contributing to extend the current understanding of cancer epigenomes and enabling the identification of clinically useful tumor biomarkers.

Highlights

The introduction of pathology tissue-chromatin immunoprecipitation (PAT-ChIP), a technique allowing chromatin immunoprecipitation (ChIP) from formalin-fixed paraffin-embedded (FFPE) tissues, has extended the application of chromatin studies to clinical patient samples
We recently introduced a new ChIP technique, named pathology tissue-chromatin immunoprecipitation (PAT-ChIP) that enables chromatin extraction and immunoprecipitation from formalin-fixed paraffin-embedded (FFPE) tissues, allowing the exploitation of a vast number of clinically annotated tissue resources stored in pathology archives [12, 13]
Limited reversal of crosslinking increases the amount of soluble chromatin isolated from FFPE samples at different times of fixation attempts to standardize the times of fixation to 24 h/48 h for most tissue types, samples derived from surgery are in most cases still crosslinked with 3.7–4% FA for times usually ranging between 24 and 72 h [25,26,27]

Summary

Introduction

The introduction of pathology tissue-chromatin immunoprecipitation (PAT-ChIP), a technique allowing chromatin immunoprecipitation (ChIP) from formalin-fixed paraffin-embedded (FFPE) tissues, has extended the application of chromatin studies to clinical patient samples. The reduced efficiency in chromatin extraction from over-fixed formalin archival samples is the main hurdle to overcome, especially when low abundant epigenetic marks (e.g., H3K4me3) are investigated. In these years, immense developments are occurring in the fields of early cancer detection, biomarker-based treatment selection, and disease response to treatments. We recently introduced a new ChIP technique, named pathology tissue-chromatin immunoprecipitation (PAT-ChIP) that enables chromatin extraction and immunoprecipitation from formalin-fixed paraffin-embedded (FFPE) tissues, allowing the exploitation of a vast number of clinically annotated tissue resources stored in pathology archives [12, 13]. The technique has been applied by several investigators [15,16,17,18,19,20,21,22,23] giving new impetus to chromatin studies in patient samples and to the identification of new potential epimarkers in function of the clinical information of patients

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