EGFR transcription in non‐small‐cell lung cancer tumours can be revealed in ctDNA by cell‐free chromatin immunoprecipitation (cfChIP)

Abstract

Determination of tumour-specific transcription based on liquid biopsies possesses a large diagnostic and prognostic potential in non-small cell lung cancer (NSCLC). Cell-free DNA (cfDNA) packed in nucleosomes mirrors the histone modification profiles present in the cells of origin. H3 lysine 36 trimethylation (H3K36me3)-modified nucleosomes are associated with active genes and, therefore, cell-free chromatin immunoprecipitation (cfChIP) of H3K36me3-associated cfDNA has the potential to delineate whether transcription of a particular gene is occurring in the cells from which its cfDNA originates. We hypothesized that cfChIP can delineate transcriptional status of genes harbouring somatic cancer mutations and analysed the recurrently observed EGFR-L858R mutation as an example. In representative NSCLC cell lines, the relationship between wild type (WT) and mutated EGFR transcriptional activity and mRNA expression levels was analysed using H3K36me3 ChIP and EGFR mRNA reverse transcription quantitative PCR (RT-qPCR), respectively. The ChIP analysis showed that both WT and mutated EGFR are transcribed, and that mRNA is similarly expressed per EGFR copy. Based on this observation, we proceeded with EGFR cfChIP using blood plasma from NSCLC patients harbouring the EGFR-L858R mutation. EGFR-WT fragments can originate from both non-tumour cells with no or low EGFR transcription and tumour cells with active EGFR transcription, whereas EGFR-L858R fragments must specifically originate from tumour cells. H3K36me3 cfChIP followed by droplet digital PCR (ddPCR) revealed significantly higher enrichment of EGFR-L858R compared to EGFR-WT fragments. This is in alignment with EGFR-L858R being actively transcribed in the NSCLC tumour cells. This study is proof-ofprinciple that cfChIP can be used to identify tumour-specific transcriptional activity of mutated alleles, which can expand the utility of liquid-biopsy-based cfDNA analyses to enhance tumour diagnostics and therapeutics.

Genome-wide epigenetic and mRNA-expression profiling followed by CRISPR/Cas9-mediated gene-disruptions corroborate the MIR141/MIR200C-ZEB1/ZEB2-FGFR1 axis in acquired EMT-associated EGFR TKI-resistance in NSCLC cells

“…Whereas cfDNA-methylation has been widely addressed, recent published results have also revealed that quantitative analyses of histone modifications using cfChIP possess a cancer diagnostic potential (53)(54)(55). The hereby-presented data points that quantifying H3K36me3 at cf nucleosomes from the basis of liquid biopsies could extend the number of genes for which it will be possible to derive tumor geneexpression information beyond what will be possible with DNA-methylation analyses.…”

New Perspectives on the Importance of Cell-Free DNA Biology

“…Pathology-related changes in transcriptional programs in specific cell types could be identified with this method. In another proof-of-principle study, immunoprecipitation of H3 lysine 36 trimethylation (H3K36me3)-modified nucleosomes, a histone modification associated with active gene transcription, was used as a liquid biopsy marker to identify tumor-specific transcriptional activity of mutated alleles in non-small cell lung cancer [ 171 , 183 ].…”

Tracing the Origin of Cell-Free DNA Molecules through Tissue-Specific Epigenetic Signatures

“…Colorectal cancer was detected with high sensitivity using cfChIP-seq and could identify subgroups of patients with distinct cancer-related transcriptional programs [ 28 ]. Another study employing blood plasma cfChIP revealed that H3K36me3 cfChIP followed by droplet digital PCR can be used to identify tumor-specific transcriptional activity of the mutated EGFR-L858R allele in non-small cell lung cancer [ 145 ]. This focus on tumor-specific transcriptional activity of genes harboring somatic mutations will help to gain more insights on the relevance of mutations in, e.g., therapy resistance mechanisms.…”