179P Immunoprecipitation of tumor-associated ctDNA fragments for novel lung cancer liquid biopsy

CTCF transcription factor binding is altered in cancer including both loss and gain of CTCF binding site occupancy. We describe a novel method for ctDNA analysis in which tumor derived plasma cell free CTCF-DNA (cfCTCF-DNA) fragments derived from cancer associated CTCF gain of occupancy binding sites, are isolated from all non-tumor plasma nucleoproteins comprising cfDNA of the same sequences by Chromatin ImmunoPrecipitation (ChIP). We developed a ChIP-Seq method for isolating and sequencing plasma cfCTCF-DNA nucleoproteins. Anti-CTCF ChIP-Seq was performed on 4 patients diagnosed with Acute Myeloid Leukaemia (AML), 5 patients with inflammatory conditions and 5 healthy volunteers. We identified 29 cfCTCF-DNA cancer associated gain of occupancy binding site sequences that were present in the ChIP isolates of cancer patients, but not present in isolates from healthy subjects or subjects with inflammatory conditions. We next developed qPCR assays for 10 of the 29 identified gain of occupancy CTCF binding sites. The 10 qPCR assays were investigated in ChIP/PCR plasma assays for detection of AML and lung cancer in a preliminary proof-of-concept study. ChIP isolates from plasma samples obtained from AML patients (n=31), lung cancer patients (n=10) and from control subjects that were either healthy (n=35), or had an inflammatory condition (n=15) were tested by qPCR for the presence of the 10 CTCF-bound binding site sequences selectively occupied in cancer. The 10 ChIP/qPCR assays were effective for detection of AML. A single qPCR assay detected 19 of 31 AML cases (61%) using a simple +/- cutoff with 1 false positive result among 50 control samples (98% specificity). Some qPCR assays were also positive for solid cancers including 4 of 10 lung cancer cases with 96% specificity using a panel of 3 qPCR assays. cfCTCF-DNA binding site occupancy biomarkers represent a new class of untapped cancer biomarkers. Further discovery studies using lung cancer samples rather than AML samples are required to identify lung cancer specific markers followed by clinical studies to ascertain clinical accuracy.