Direct analysis of transcription factor protected cfDNA in plasma by ChIP-seq: Measurement of altered CTCF binding in cancer is a novel biomarker for liquid biopsy

Background The aggregate presence of cell free CTCF-DNA (cfCTCF-DNA) nucleoproteins in plasma has been reported. But the occupancy of individual CTCF binding sites in plasma cfCTCF-DNA has not been studied. Results For the first time, we have isolated endogenous plasma cfCTCF-DNA by chromatin immunoprecipitation (ChIP) and sequenced the associated cfDNA (ChIP-Seq) in samples from cancer patients and healthy subjects. The mean observed enrichment of plasma cfCTCF-DNA by ChIP was 180-fold with a mean CTCF recovery of 48%. Background cfDNA, primarily in the form of nucleosomes, was almost completely physically removed (> 99.7%) by ChIP and the remainder was removed bioinformatically. cfCTCF-DNA sequences were partitioned into low or high affinity binding cfCTCF-DNA nucleoproteins in silico , on the basis of binding motif concordance with the consensus CTCF binding motif. We observed that cfCTCF-DNA was quantitatively and qualitatively different in samples from healthy subjects or cancer patients. High affinity cfCTCF-DNA nucleoproteins were present only in the plasma of cancer patients and not in samples from healthy subjects. Testing of small developmental and validation sample cohorts for ChIP-seq peaks for a 14 member panel of high affinity CTCF loci sequences, showed excellent specificity and sensitivity for cancer detection. Conclusions Plasma sequence data sets comprising > 99% pure CTCF protected ctDNA can be prepared using a 2-step process involving the physical removal of most background cfDNA in vitro , followed by removal of all remaining background cfDNA in silico . CTCF plasma ChIP-seq represents a novel liquid biopsy method for a novel class of liquid biopsy biomarkers.