205P - A novel immunoprecipitation/PCR method for detection of plasma cfDNA fragments selectively occupied by CTCF in cancer

Background

CTCF-binding site (BS) occupancy is plastic, tissue specific and altered in cancer. This principle underlies some fragmentomics methods. We hypothesized that ChIP-Seq of plasma CTCF-cfDNA might identify CTCF-BSs selectively occupied in cancer and that, having removed all nucleosomes or other non-CTCF bound cfDNA by CTCF immunoprecipitation (IP), the presence of cfDNA fragments corresponding to sites selectively CTCF occupied in cancer may be tested for by PCR. We now report a novel liquid biopsy method, and initial results, based on this principle.

Methods

(i) Identification of CTCF-BSs selectively occupied in cancer samples. Plasma samples were collected from healthy volunteers (n=5), and patients with inflammatory conditions (n=5) or cancer (n=5). IP was performed using anti-CTCF coated magnetic beads and immunoprecipitated cfDNA was extracted, amplified and sequenced on an Illumina platform. (ii) We developed specific qPCR assays for 10 CTCF-BS sequences that we observed to be preferentially bound in the plasma of cancer patients vs either inflammatory conditions or healthy subjects. The panel of qPCR assays was tested on CTCF associated cfDNA isolated from plasma samples (1ml) obtained from patients diagnosed with a variety of common solid cancer diseases (n=48).

Results

We observed that specific CTCF IP resulted in isolation of cfDNA fragments with a frequency peak in the 30-80bp range that was not present in negative controls (mouse-IgG). These short cfDNA fragments included previously described CTCF binding sites. Moreover, CTCF binding sites were not randomly distributed on cfDNA fragments but located at the centre of each fragment. Among the CTCF occupied binding sites observed, we identified 29 sites that were significantly differentially bound in cancer. We observed highly sensitive and specific qPCR test results for a number of common solid cancer diseases including high sensitivity at disease stage I and II.

Conclusions

This is the first demonstration of IP of plasma CTCF-cfDNA. IP followed by multiplex PCR may form the basis of a rapid, low cost liquid biopsy method that obviates expensive library preparation and NGS.

Clinical trial identification

Editorial acknowledgement

Legal entity responsible for the study

Belgian Volition.

Funding

Belgian Volition.

Disclosure

D. Pamart, J-V. Turatsinze, B. Cuvelier, J-V. Turatsinze: Financial Interests, Personal, Full or part-time Employment: Belgian Volition. M. Herzog: Financial Interests, Personal, Full or part-time Employment: Belgian Volition; Financial Interests, Personal, Stocks/Shares: VolitionRx. J. Micallef: Financial Interests, Personal, Officer, Chief Scientific Officer: VolitionRx Ltd; Financial Interests, Personal, Stocks/Shares: VolitionRx Ltd.