Cancer Methylation Biomarkers in Circulating Cell-Free DNA

Pidsley, Ruth; Stirzaker, Clare

doi:10.1007/978-981-13-8958-0_10

Cited by 3 publications

(2 citation statements)

References 142 publications

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“…For example, Septin9 gene ( SEPT9 ) hypermethylation frequently occurs in CRC and may serve as a universal biomarker for CRC monitoring ( 15 ). Indeed, DNA methylation biomarkers have shown potential in tumor screening, prognosis, therapeutic efficacy evaluation, and personalized treatment ( 16 , 17 ).…”

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confidence: 99%

Efficient detection and post-surgical monitoring of colon cancer with a multi-marker DNA methylation liquid biopsy

Jin

Zhu

Shao

et al. 2021

Proc. Natl. Acad. Sci. U.S.A.

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Multiplex assays, involving the simultaneous use of multiple circulating tumor DNA (ctDNA) markers, can improve the performance of liquid biopsies so that they are highly predictive of cancer recurrence. We have developed a single-tube methylation-specific quantitative PCR assay (mqMSP) that uses 10 different methylation markers and is capable of quantitative analysis of plasma samples with as little as 0.05% tumor DNA. In a cohort of 179 plasma samples from colorectal cancer (CRC) patients, adenoma patients, and healthy controls, the sensitivity and specificity of the mqMSP assay were 84.9% and 83.3%, respectively. In a head-to-head comparative study, the mqMSP assay also performed better for detecting early-stage (stage I and II) and premalignant polyps than a published SEPT9 assay. In an independent longitudinal cohort of 182 plasma samples (preoperative, postoperative, and follow-up) from 82 CRC patients, the mqMSP assay detected ctDNA in 73 (89.0%) of the preoperative plasma samples. Postoperative detection of ctDNA (within 2 wk of surgery) identified 11 of the 20 recurrence patients and was associated with poorer recurrence-free survival (hazard ratio, 4.20; P = 0.0005). With subsequent longitudinal monitoring, 14 patients (70%) had detectable ctDNA before recurrence, with a median lead time of 8.0 mo earlier than seen with radiologic imaging. The mqMSP assay is cost-effective and easily implementable for routine clinical monitoring of CRC recurrence, which can lead to better patient management after surgery.

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confidence: 99%

Efficient detection and post-surgical monitoring of colon cancer with a multi-marker DNA methylation liquid biopsy

Jin

Zhu

Shao

et al. 2021

Proc. Natl. Acad. Sci. U.S.A.

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“…Further, the MBPS assay can measure cfDNA methylation. This is of great clinical interest as cfDNA in human blood can serve as a liquid biopsy to provide a minimally invasive method for predictive and prognostic marker detection [34]. Levels of cfDNA are generally very low, ranging from~0 to 50 ng/ml blood, and the isolated cfDNA is commonly~170-300 bp, mostly corresponding to~170 bp mono-nucleosomal and~300 bp di-nucleosomal DNA fragments.…”

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confidence: 99%

Comprehensive evaluation of targeted multiplex bisulphite PCR sequencing for validation of DNA methylation biomarker panels

et al. 2020

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Background: DNA methylation is a well-studied epigenetic mark that is frequently altered in diseases such as cancer, where specific changes are known to reflect the type and severity of the disease. Therefore, there is a growing interest in assessing the clinical utility of DNA methylation as a biomarker for diagnosing disease and guiding treatment. The development of an accurate loci-specific methylation assay, suitable for use on low-input clinical material, is crucial for advancing DNA methylation biomarkers into a clinical setting. A targeted multiplex bisulphite PCR sequencing approach meets these needs by allowing multiple DNA methylated regions to be interrogated simultaneously in one experiment on limited clinical material. Results: Here, we provide an updated protocol and recommendations for multiplex bisulphite PCR sequencing (MBPS) assays for target DNA methylation analysis. We describe additional steps to improve performance and reliability: (1) pre-sequencing PCR optimisation which includes assessing the optimal PCR cycling temperature and primer concentration and (2) post-sequencing PCR optimisation to achieve uniform coverage of each amplicon. We use a gradient of methylated controls to demonstrate how PCR bias can be assessed and corrected. Methylated controls also allow assessment of the sensitivity of methylation detection for each amplicon. Here, we show that the MBPS assay can amplify as little as 0.625 ng starting DNA and can detect methylation differences of 1% with a sequencing coverage of 1000 reads. Furthermore, the multiplex bisulphite PCR assay can comprehensively interrogate multiple regions on 1-5 ng of formalin-fixed paraffin-embedded DNA or circulating cell-free DNA. Conclusions: The MBPS assay is a valuable approach for assessing methylated DNA regions in clinical samples with limited material. The optimisation and additional quality control steps described here improve the performance and reliability of this method, advancing it towards potential clinical applications in biomarker studies.

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Transfer of blocker-based qPCR reactions for DNA methylation analysis into a microfluidic LoC system using thermal modeling

et al. 2022

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Changes in the DNA methylation landscape are associated with many diseases like cancer. Therefore, DNA methylation analysis is of great interest for molecular diagnostics and can be applied, e.g., for minimally invasive diagnostics in liquid biopsy samples like blood plasma. Sensitive detection of local de novo methylation, which occurs in various cancer types, can be achieved with quantitative HeavyMethyl-PCR using oligonucleotides that block the amplification of unmethylated DNA. A transfer of these quantitative PCRs (qPCRs) into point-of-care (PoC) devices like microfluidic Lab-on-Chip (LoC) cartridges can be challenging as LoC systems show significantly different thermal properties than qPCR cyclers. We demonstrate how an adequate thermal model of the specific LoC system can help us to identify a suitable thermal profile, even for complex HeavyMethyl qPCRs, with reduced experimental effort. Using a simulation-based approach, we demonstrate a proof-of-principle for the successful LoC transfer of colorectal SEPT9/ ACTB-qPCR from Epi Procolon® colorectal carcinoma test, by avoidance of oligonucleotide interactions.

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Cancer Methylation Biomarkers in Circulating Cell-Free DNA

Cited by 3 publications

References 142 publications

Efficient detection and post-surgical monitoring of colon cancer with a multi-marker DNA methylation liquid biopsy

Efficient detection and post-surgical monitoring of colon cancer with a multi-marker DNA methylation liquid biopsy

Comprehensive evaluation of targeted multiplex bisulphite PCR sequencing for validation of DNA methylation biomarker panels

Transfer of blocker-based qPCR reactions for DNA methylation analysis into a microfluidic LoC system using thermal modeling

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