Abstract 2428: Novel ctDNA technology for early cancer detection by immunoprecipitation of tumor associated ctDNA fragments and analysis by qPCR
Authors: Micallef Jacob , Pamart Dorian , Turatsinze Jean-Valery V. , Bygott Thomas , Berman Benjamin , Cuvelier Brieuc , Herzog Marielle 
Published: March 2024

Abstract
Introduction: Current ctDNA methods involve extraction of all plasma cfDNA, next generation sequencing (NGS) and complex bioinformatic analysis. We have developed a novel ctDNA analysis in which classes of tumor derived ctDNA are isolated, with removal of all non-tumor cfDNA of the same sequences, for analysis by qPCR. We now report the results of this analysis on cohorts of patients diagnosed with common solid cancers.
Methods: We developed a chromatin immunoprecipitation (ChIP)/qPCR method for the isolation of cell free plasma CTCF-DNA (cfCTCF-DNA) nucleoproteins that include binding sites selectively occupied in cancer. CfDNA fragments of non-cancer origin that comprise the same CTCF binding site sequences are nucleosome covered and washed away. We performed plasma CTCF ChIP-Seq discovery experiments to identify CTCF gain of occupancy cancer biomarker sequences by analysis of plasma samples taken from cancer patients as well as patients with inflammatory conditions and healthy volunteers. Suitable pan-cancer, or cancer tissue specific, biomarkers were selected as characterized CTCF binding site sequences that were present in the ChIP isolates of cancer patients but absent in isolates from patients without cancer. We then developed qPCR assays for selected CTCF gain of occupancy biomarker sequences. We initially investigated whether CTCF gain of occupancy biomarkers would identify patients with cancer in a small cohort of subjects with acute myeloid leukemia (AML) (n=31), breast (n=10), prostate (n=10), liver (n=10) or colorectal cancer (CRC) (n=13) and 50 control subjects including 15 subjects with an inflammatory condition.
Results: In our small initial experiments, a simple cutoff where a qPCR result exceeding the cutoff was positive, a single qPCR assay detected 19 of 31 AML cases (61%) with 1 false positive result among 50 control samples (98% specificity). Addition of a second qPCR assay to make a 2-member qPCR panel, where a result exceeding cutoff in either or both assays was classified as positive, resulted in the detection of 23 of 31 AML cases (74%) with 2 false positive results (96% specificity). On the same basis a 2-member qPCR panel also detected 58% of solid cancers including 44% of stage I cases. Another 2-member qPCR panel detected 77% of CRC cases at 92% specificity. We now report the results of investigations of larger cohorts of patients with common solid cancers by cancer type, stage and tissue specificity.
Conclusion: Combining the isolation of plasma cfCTCF-DNA nucleoproteins with PCR analysis of cancer associated CTCF gain of occupancy binding site sequences is a novel ctDNA analysis technology that may provide the basis of useful, rapid, low cost, automated liquid biopsy methods. Further clinical studies to ascertain the clinical accuracy of the method are required.
Citation Format: Jacob Micallef, Dorian Pamart, Jean-Valery V. Turatsinze, Thomas Bygott, Benjamin Berman, Brieuc Cuvelier, Marielle Herzog. Novel ctDNA technology for early cancer detection by immunoprecipitation of tumor associated ctDNA fragments and analysis by qPCR [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2428.