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5-hydroxymethylated biomarkers in cell-free DNA predict occult colorectal cancer up to 36 months prior to diagnosis in the PLCO Cancer Screening Trial

ABSTRACT: Purpose: Using the Prostate, Lung, Colorectal and Ovarian (PLCO) Cancer Screening Trial samples, we identified cell-free DNA (cfDNA) biomarker candidate genes bearing the epigenetic mark 5-hydroxymethylcytosine (5hmC) that detect occult colorectal cancer (CRC) up to 36 mo prior to clinical diagnosis. Methods: PLCO study subjects were matched by age, race, and sex as cases (n = 201, diagnosed with CRC within 36 mo of blood collection) and controls (n = 402, no cancer diagnosis on follow-up, average 16.3 years after entering the study). Archived plasma samples (300 µL per study subject) were obtained from the National Cancer Institute (NCI), and we employed the sensitive 5hmC-Seal chemical labeling approach on 3 - 8 ng of extracted cfDNA. Following next-generation sequencing (NGS) and genome-wide mapping of 5hmC, we then conducted association studies and machine-learning modeling to analyze the genome-wide 5hmC profiles within training and validation groups that were randomly selected at a 2:1 ratio. Results: Robust genome-wide 5hmC profiles were successfully obtained from these decades-old samples. Association analyses using the Cox proportional hazards models suggested several epigenetic pathways relevant to CRC development distinguishing cases from controls. A weighted Cox model, comprised of 32-associated gene bodies, showed predictive detection value for CRC as early as 24-36 mo prior to overt tumor diagnosis. Furthermore, a trend for increased predictive power was observed for blood samples collected closer to CRC diagnosis. Notably, the 5hmC-based predictive model showed comparable performance regardless of sex and self-reported race/ethnicity, and significantly outperformed risk factors such as age and obesity assessed as BMI (body mass index). Conclusion: An assay and machine learning modeling of 5hmC epigenetic signals on cfDNA revealed candidate biomarkers and a scoring algorithm with the potential to predict CRC occurrence despite the absence of clinical symptoms or the availability of effective predictors. Developing a minimally-invasive clinical assay that detects 5hmC-modified biomarkers holds promise for improving early CRC detection and ultimately patient outcomes. Future investigations to expand this strategy to prospectively collected samples are warranted.

ORGANISM(S): Homo sapiens

PROVIDER: GSE263030 | GEO | 2025/01/22

REPOSITORIES: GEO

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Project description:Background: For the majority of patients of metastatic colorectal cancer (mCRC), systematic chemotherapy has been a choice for palliative treatment. When first-line chemotherapies do not work, targeted therapies are administered. Because of inter-individual genetic and epigenetic variations, patients with mCRC who are treated with these therapies may respond differently. It is of great clinical interest to elucidate response mechanisms that could be utilized in predicting individual response to these treatments. Methods: A highly sensitive technique, the 5hmC-Seal was applied to profile genome-wide 5-hydroxymethylcytosines (5hmC) in 155 cfDNA samples from patients with mCRC recruited for two clinical trials at Zhongshan Hospital, Fudan University in China. Response to systematic chemotherapy and targeted therapy (becacizumab or cetuximab) (CR, complete response; PR, partial response; SD, stable disease; PD, progressive disease) was measured and a 5hmC-based predictive model in cfDNA was developed using a machine learning approach in the PROVE Study (NCT03679039) (n = 100), followed by validation in the METHOD Study (NCT03599947) (n = 55). Results: Genome-wide mapping of 5hmC in patient-derived cfDNA suggested a distinct 5hmC landscape showing tissue origin and gene regulatory relevance. A eleven-gene model involving relevant pathways such as Rap1 signaling pathway, cAMP signaling pathway, Phospholipase D signaling pathway and cGMP-PKG signaling pathway, distinguished patients with mCRC who responded to chemotherapy, regardless of targeted therapy, resectability, or mutation status in the validation samples (AUC=0.77; 95% CI, 0.64-0.90), outperforming RAS mutation status (AUC=0.70; 95% CI, 0.57-0.83). In addition, the 5hmC model also showed a trend of capacity for predicting progression-free survival (PFS) for mCRC. Conclusions: Genome-wide mapping reflected relevant pathways and functional interaction networks implicated in the determination of treatment response for patients with mCRC. The 5hmC-Seal model in cfDNA provided a promising non-invasive method for predicting response to systematic chemotherapy in patients with mCRC, who will be essential for personalized medicine.

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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5-hydroxymethylated biomarkers in cell-free DNA predict occult colorectal cancer up to 36 months prior to diagnosis in the PLCO Cancer Screening Trial

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