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Heat selection enables highly scalable methylome profiling in cell-free DNA for noninvasive monitoring of cancer patients

ABSTRACT: Genome-wide analysis of cell-free DNA (cfDNA) methylation profile has been recognized as a promising approach for sensitive and specific detection of many cancers. However, scaling such genome-wide assays for clinical translation is impractical due to the high cost of whole genome bisulfite sequencing. We have shown that the small fraction of GC-rich genome is highly enriched in CpG sites and disproportionately harbors the majority of cancer-specific methylation signature. Here, we report on the simple but effective Heat enrichment of CpG-rich regions for Bisulfite Sequencing (Heatrich-BS) platform that allows for focused methylation profiling in these highly informative regions. Our novel method and bioinformatics algorithm enable accurate tumor burden estimation with high sensitivity and quantitative tracking of colorectal cancer patientÃ¢Â€Â™s response to treatment, at much reduced sequencing cost suitable for frequent monitoring. We also show, for the first time, tumor epigenetic subtyping from cfDNA using Heatrich-BS, which could enable patient stratification from non-invasive liquid biopsy. As such, Heatrich-BS holds great potential for highly scalable screening and regular monitoring of cancer using liquid biopsy.

PROVIDER: EGAS00001006198 | EGA |

REPOSITORIES: EGA

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Project description:Background: Liquid biopsy based on cell free DNA (cfDNA) analysis holds significant promise as a minimally invasive approach for the diagnosis, genotyping and monitoring of solid malignancies. Human tumors release cfDNA in the bloodstream through a combination of events, including cell death, active and passive release. However, the precise mechanisms leading to cfDNA shedding remain to be characterized. Addressing this question in patients is confounded by several factors, such as tumor burden extent, anatomical and vasculature barriers, release of nucleic acids from normal cells. In this work, we exploited cancer models to dissect basic mechanisms of DNA release. Methods: We measured cell loss ratio, doubling time and cfDNA release in the supernatant of a colorectal cancer (CRC) cell line collection (N=76) representative of the molecular subtypes previously identified in cancer patients. Association analyses between quantitative parameters of cfDNA release, cell proliferation and molecular features were evaluated. Functional experiments were performed to test the impact of modulating DNA methylation on cfDNA release. Results: Higher levels of supernatant cfDNA were significantly associated with slower cell cycling and increased cellular crashes. In addition, a higher cfDNA shedding was found in non-CpG Island Methylator Phenotype (CIMP) models. These results indicate a positive correlation between lower methylation and increased cfDNA levels. To explore this further, we exploited methylation microarrays to identify a subset of probes significantly associated with cfDNA shedding and derive a methylation signature capable of discriminating high from low cfDNA releasers. We applied this signature to an independent set of 176 CRC cell lines and patient derived organoids to select 14 models predicted to be low or high releasers. The methylation profile successfully predicted the amount of cfDNA released in the supernatant. At the functional level, genetic ablation of DNA methyl-transferases increased chromatin accessibility and DNA fragmentation, leading to increased cfDNA release in isogenic CRC cell lines. Furthermore, in vitro treatment of five low releaser CRC cells with a demethylating agent was able to induce a significant increase in cfDNA shedding. Conclusion: Methylation status of cancer cell lines contributes to the variability of cfDNA shedding in vitro. Changes in methylation pattern are associated with cfDNA release levels and might be exploited to increase sensitivity of liquid biopsy assays.

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Heat selection enables highly scalable methylome profiling in cell-free DNA for noninvasive monitoring of cancer patients

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