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

BACKGROUND: 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. 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. RESULTS: We 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. CONCLUSIONS: EPAT-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.

PAT-ChIP coupled with laser microdissection allows the study of chromatin in selected cell populations from paraffin-embedded patient samples.

BACKGROUND: The recent introduction of pathology tissue-chromatin immunoprecipitation (PAT-ChIP), a technique allowing chromatin immunoprecipitation from formalin-fixed and paraffin-embedded (FFPE) tissues, has expanded the application potential of epigenetic studies in tissue samples. However, FFPE tissue section analysis is strongly limited by tissue heterogeneity, which hinders linking the observed epigenetic events to the corresponding cellular population. Thus, ideally, to take full advantage of PAT-ChIP approaches, procedures able to increase the purity and homogeneity of cell populations from FFPE tissues are required. RESULTS: In this study, we tested the use of both core needle biopsies (CNBs) and laser microdissection (LMD), evaluating the compatibility of these methods with the PAT-ChIP procedure. Modifications of the original protocols were introduced in order to increase reproducibility and reduce experimental time. We first demonstrated that chromatin can be prepared and effectively immunoprecipitated starting from 0.6-mm-diameter CNBs. Subsequently, in order to assess the applicability of PAT-ChIP to LMD samples, we tested the effects of hematoxylin or eosin staining on chromatin extraction and immunoprecipitation, as well as the reproducibility of our technique when using particularly low quantities of starting material. Finally, we carried out the PAT-ChIP using chromatin extracted from either normal tissue or neoplastic lesions, the latter obtained by LMD from FFPE lung sections derived from mutant K-ras(v12) transgenic mice or from human adeno- or squamous lung carcinoma samples. Well characterized histone post-translational modifications (HPTMs), such as H3K4me3, H3K27me3, H3K27Ac, and H3K9me3, were specifically immunoselected, as well as the CTCF transcription factor and RNA polymerase II (Pol II). CONCLUSIONS: Epigenetic profiling can be performed on enriched cell populations obtained from FFPE tissue sections. The improved PAT-ChIP protocol will be used for the discovery and/or validation of novel epigenetic biomarkers in FFPE human samples.