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Tissue-specific cell-free DNA degradation quantifies circulating tumor DNA burden

ABSTRACT: Profiling of circulating tumor DNA (ctDNA) may offer a non-invasive approach to monitor disease progression. Here, we developed a quantitative method, exploiting local tissue-specific cell-free DNA (cfDNA) degradation patterns, that accurately estimates ctDNA burden independent of genomic aberrations. Nucleosome-dependent cfDNA degradation at promoters and first exon-intron junctions was strongly associated with differential transcriptional activity in tumors and blood. A quantitative model, based on just 6 regulatory regions, could accurately predict ctDNA levels in colorectal cancer patients. Strikingly, a model restricted to blood-specific regulatory regions could predict ctDNA levels across both colorectal and breast cancer patients.

PROVIDER: EGAS00001004657 | EGA |

REPOSITORIES: EGA

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Tissue-specific cell-free DNA degradation quantifies circulating tumor DNA burden.

Zhu Guanhua G Guo Yu A YA Ho Danliang D Poon Polly P Poh Zhong Wee ZW Wong Pui Mun PM Gan Anna A Chang Mei Mei MM Kleftogiannis Dimitrios D Lau Yi Ting YT Tay Brenda B Lim Wan Jun WJ Chua Clarinda C Tan Tira J TJ Koo Si-Lin SL Chong Dawn Q DQ Yap Yoon Sim YS Tan Iain I Ng Sarah S Skanderup Anders J AJ

Nature communications 20210413 1

Profiling of circulating tumor DNA (ctDNA) may offer a non-invasive approach to monitor disease progression. Here, we develop a quantitative method, exploiting local tissue-specific cell-free DNA (cfDNA) degradation patterns, that accurately estimates ctDNA burden independent of genomic aberrations. Nucleosome-dependent cfDNA degradation at promoters and first exon-intron junctions is strongly associated with differential transcriptional activity in tumors and blood. A quantitative model, based ...[more]

PMID: 33850132

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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.

Project description:Colorectal cancer (CRC) is one of the most common gastrointestinal tumors. According to the latest cancer report, the incidence and mortality rates of CRC are both ranked top 5 among malignant tumors worldwide and continue to rise. Patients who receive treatment in the early stage (stage I) have a 5-year survival rate of approximately 90%. However, for high-risk stage II and III colorectal cancer patients, the 5-year survival rate is only 40%-70%, and almost half of the patients experience postoperative recurrence and metastasis. Circulating tumor DNA (ctDNA) is a small fraction of total cell-free DNA (cfDNA) in peripheral blood circulation, carrying tumor-specific genetic and epigenetic information. It can usually be detected in the serum or plasma of tumor patients in peripheral blood. Studies have shown that methylation detection of plasma ctDNA can be used for predicting the efficacy and prognosis of tumor postoperatively, as well as for dynamic monitoring. Current methods for monitoring CRC recurrence include testing for carcinoembryonic antigen (CEA) in blood and periodic computed tomography (CT) scans. However, due to the low sensitivity of CEA and the radiation and cost limitations of CT examination, the disease status of postoperative CRC patients cannot be well-monitored. ctDNA is a promising biomarker for monitoring the recurrence and metastasis of CRC. Research results have shown that ctDNA can be detected in all subjects before surgery, and the changes in ctDNA levels are related to the extent of surgical resection. The detection of ctDNA after surgery generally indicates recurrence within one year. ctDNA may be a more reliable and sensitive indicator than the current standard biomarker CEA, providing a window for early intervention. This multicenter, prospective, and randomized controlled cohort study uses a single-tube methylation-specific quantitative PCR (mqMSP) detection, which detects 10 different methylation markers and can quantitatively analyze plasma samples containing tumor DNA as low as 0.05%. This study will use the ctDNA methylation detection technology to conduct quantitative detection of ctDNA methylation in the plasma of enrolled patients, hoping to predict the recurrence and metastasis risk of patients at an earlier stage through ctDNA changes, and to explore the value of ctDNA detection in guiding postoperative follow-up for high-risk stage II and III CRC.

Dataset Information

Tissue-specific cell-free DNA degradation quantifies circulating tumor DNA burden

Publications

Tissue-specific cell-free DNA degradation quantifies circulating tumor DNA burden.

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