EGFR transcription in non-small-cell lung cancer tumours can be revealed in ctDNA by cell-free chromatin immunoprecipitation (cfChIP).
Christoffer Trier MånssonJohan Vad-NielsenPeter MeldgaardAnders Lade NielsenBoe Sandahl SorensenPublished in: Molecular oncology (2021)
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 nontumour 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-of-principle 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.
Keyphrases
- small cell lung cancer
- epidermal growth factor receptor
- tyrosine kinase
- induced apoptosis
- transcription factor
- advanced non small cell lung cancer
- cell free
- gene expression
- cell cycle arrest
- brain metastases
- genome wide
- dna methylation
- risk assessment
- squamous cell carcinoma
- cell death
- copy number
- oxidative stress
- young adults
- endoplasmic reticulum stress
- cell proliferation
- lymph node metastasis
- peritoneal dialysis
- patient reported
- chronic kidney disease
- fine needle aspiration
- molecularly imprinted
- amino acid