Applications in DNA Methylation Analysis

Simultaneous analysis of mutations and methylations in circulating cell-free DNA for hepatocellular carcinoma detection.

Pei Wang, Qianqian Song, Jie Ren, Weilong Zhang, Yuting Wang, Lin Zhou, Dongmei Wang, Kun Chen, Liping Jiang, Bochao Zhang, Wanqing Chen, Chunfeng Qu, Hong Zhao, Yuchen Jiao. National Cancer Center of China. Science Translational Medicine 14, eabp8704 (2022) 23 November 2022

Abstract Cell-free DNA (cfDNA)–based liquid biopsy is a promising approach for the early detection of cancer. A major hurdle is the limited yield of cfDNA from one blood draw, limiting the use of most samples to one test of either mutation or methylation. Here, we develop a technology, Mutation Capsule Plus (MCP), which enables multiplex profiling of one cfDNA sample, including simultaneous detection of genetic and epigenetic alterations and genome-wide discovery of methylation markers. With this technology, we performed de novo screening of methylation markers on cfDNA samples from 30 hepatocellular carcinoma (HCC) cases and 30 non-HCC controls. The methylation markers enriched in HCC cfDNA were further profiled in parallel with a panel of mutations on a training cohort of 60 HCC and 60 non-HCC cases, resulting in an HCC detection model. We validated the model in an independent retrospective cohort with 58 HCC and 198 non-HCC cases and got 90% sensitivity with 94% specificity. Furthermore, we applied the model to a prospective cohort of 311 asymptomatic hepatitis B virus carriers with normal liver ultrasonography and serum AFP concentration. The model detected four of the five HCC cases in the cohort, showing 80% sensitivity and 94% specificity. These findings demonstrate that the MCP technology has potential for the discovery and validation of multiomics biomarkers for the noninvasive detection of cancer. This study also provides a comprehensive database of genetic and epigenetic alterations in the cfDNA of a large cohort of HCC cases and high-risk non-HCC individuals.

(Methods Section) cfDNA was extracted from the plasma samples using the Apostle MiniMax cfDNA isolation kit (C43468, Apostle). 

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

Jin et al. PNAS February 2, 2021 118 (5) e2017421118; https://doi.org/10.1073/pnas.2017421118 

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. 

Plasma DNA extraction was performed using 2 to 5 mL of plasma with the Apostle MiniMax High-Efficiency cfDNA Isolation Kit, according to the product manual. 

A systematic evaluation of stool DNA preparation protocols for colorectal cancer screening via analysis of DNA methylation biomarkers.

Jin et al. Clinical Chemistry and Laboratory Medicine (CCLM). 2021; 59(1): 91–99

Objectives - Colorectal cancer (CRC) screening using stool samples is now in routine use where tumor DNA methylation analysis for leading markers such as NDRG4 and SDC2 is an integral part of the test. However, processing stool samples for reproducible and efficient extraction of human genomic DNA remains a bottleneck for further research into better biomarkers and assays.

Methods - We systematically evaluated several factors involved in the processing of stool samples and extraction of DNA. These factors include: stool processing (solid and homogenized samples), preparation of DNA from supernatant and pellets, and DNA extraction with column and magnetic beads-based methods. Furthermore, SDC2 and NDRG4 methylation levels were used to evaluate the clinical performance of the optimal protocol.

Results - The yield of total and human genomic DNA (hgDNA) was not reproducible when solid stool scraping is used, possibly due to sampling variations. More reproducible results were obtained from homogenized stool samples. Magnetic beads-based DNA extraction using the supernatant from the homogenized stool was chosen for further analysis due to better reproducibility, higher hgDNA yield, lower non-hgDNA background, and the potential for automation. With this protocol, a combination of SDC2 and NDRG4 methylation signals with a linear regression model achieved a sensitivity and specificity of 81.82 and 93.75%, respectively.

Conclusions - Through the systematic evaluation of different stool processing and DNA extraction methods, we established a reproducible protocol for analyzing tumor DNA methylation markers in stool samples for colorectal cancer screening.

(Methods section) For magnetic beads-based method, Apostle Stool gDNA Isolation Kit (APOSTLE) was used according to the manufacturer’s instructions. Either 0.2 g pellets or 0.2 mL supernatant from homogenized stool was mixed with 1 mL lysis buffer (APOSTLE) for DNA extractions.

A method for early diagnosis of lung cancer from tumor originated DNA fragments using plasma cfDNA methylome and fragmentome profiles.

 Yeo Jin Kim, Hahyeon Jeona, Sungwon Jeon, et al.  Molecular and Cellular Probes Volume 66, December 2022

Abstract Early detection is critical for minimizing mortality from cancer. Plasma cell-free DNA (cfDNA) contains the signatures of tumor DNA, allowing us to quantify the signature and diagnose early-stage tumors. Here, we report a novel tumor fragment quantification method, TOF (Tumor Originated Fragment) for the diagnosis of lung cancer by quantifying and analyzing both the plasma cfDNA methylation patterns and fragmentomic signatures. TOF utilizes the amount of ctDNA predicted from the methylation density information of each cfDNA read mapped on 6243 lung-tumor-specific CpG markers. The 6243 tumor-specific markers were derived from lung tumor tissues by comparing them with corresponding normal tissues and healthy blood from public methylation data. TOF also utilizes two cfDNA fragmentomic signatures: 1) the short fragment ratio, and 2) the 5′ end-motif profile. We used 298 plasma samples to analyze cfDNA signatures using enzymatic methyl-sequencing data from 201 lung cancer patients and 97 healthy controls. The TOF score showed 0.98 of the area under the curve in correctly classifying lung cancer from normal samples. The TOF score resolution was high enough to clearly differentiate even the early-stage non-small cell lung cancer patients from the healthy controls. The same was true for small cell lung cancer patients.

(Methods Section) Cell-free DNA was extracted from 3 to 5 ml of plasma using XXX method or Apostle MiniMax High Efficiency Isolation Kit (Beckman Coulter Life Sciences, C40603), according to the manufacturers’ procedures. 

Nicotine dose-dependent epigenomic-wide DNA methylation changes in the mice with long-term electronic cigarette exposure. 

Gang Peng,* Yibo Xi,* Chiara Bellini, Kien Pham, Zhen W Zhuang, Qin Yan, Man Jia, Guilin Wang, Lingeng Lu, Moon-Shong Tang, Hongyu Zhao, and He Wang American Journal of Cancer Research. Volume 12, Issue 8, August 2022, Pages 3679–3692. 

Abstract

Epigenomic-wide DNA methylation profiling holds the potential to reflect both electronic cigarette exposure-associated risks and individual poor health outcomes. However, a systemic study in animals or humans is still lacking. Using the Infinium Mouse Methylation BeadChip, we examined the DNA methylation status of white blood cells in male ApoE-/- mice after 14 weeks of electronic cigarette exposure with the InExpose system (2 hr/day, 5 days/week, 50% PG and 50% VG) with low (6 mg/ml) and high (36 mg/ml) nicotine concentrations. Our results indicate that electronic cigarette aerosol inhalation induces significant alteration of 8,985 CpGs in a dose-dependent manner (FDR<0.05); 7,389 (82.2%) of the CpG sites are annotated with known genes. Among the top 6 significant CpG sites (P-value<1e-8), 4 CpG sites are located in the known genes, and most (3/5) of these genes have been related to cigarette smoking. The other two CpGs are close to/associated with the Phc2 gene that was recently linked to smoking in a transcriptome-wide associations study. Furthermore, the gene set enrichment analysis highlights the activation of MAPK and 4 cardiomyocyte/cardiomyopathy-related signaling pathways (including adrenergic signaling in cardiomyocytes and arrhythmogenic right ventricular cardiomyopathy) following repeated electronic cigarette use. The MAPK pathway activation correlates well with our finding of increased cytokine mRNA expression after electronic cigarette exposure in the same batch of mice. Interestingly, two pathways related to mitochondrial activities, namely mitochondrial gene expression and mitochondrial translation, are also activated after electronic cigarette exposure. Elucidating the relationship between these pathways and the increased circulating mitochondrial DNA observed here will provide further insight into the cell-damaging effects of prolonged inhalation of e-cigarette aerosols.

(Methods section) Plasma cfDNA and mtDNA/nDNA ratio and oxidization of cell-free DNA

For measurement of in vivo plasma mtDNA/nDNA ratio, mice plasma was first subjected to cell-free DNA (cfDNA) extraction using Apostle minimax cfDNA extraction kit (ApostleBio, CA, US). 

Quantifying Fetal DNA in Maternal Blood Plasma by ddPCR Using DNA Methylation.

E. Hall, T. Riel, M. Ramesh, M. Gencoglu, O. Mikhaylichenko, R. Dannebaum, S. Margeridon, M. Herrera. Bio-Rad Laboratories, Pleasanton, CA. Association for Molecular Pathology 2022 Annual Meeting Abstracts. J Mol Diagn 2022, 24:S1 Abstract G072.

Introduction: The proportion of cell-free DNA (cfDNA) circulating in maternal blood that originates from the fetus, the fetal fraction, is an important quality control metric when performing tests on fetal-derived cfDNA. Epigenetic differences produce dissimilar DNA methylation patterns, allowing for leveraging regions of high methylation contrast using methylation-sensitive restriction enzyme (MSRE) digestion to quantify fetal and maternal DNA via droplet digital PCR (ddPCR). This advancement positions ddPCR as a faster and less expensive alternative to next-generation sequencing (NGS) for fetal fraction estimation.

Methods: Assays were designed to target MSRE- compatible regions with high methylation contrast between maternal and fetal cfDNA. Fetal assays targeted sites hypermethylated in fetal cfDNA and maternal assays targeted sites hypermethylated in maternal cfDNA. The assay multiplex was tested against contrived and clinical samples using an in-droplet MSRE-ddPCR workflow. The reaction mix was dropletized to create about 20,000 droplets per 24-μL reaction, thermocycled, and analyzed in a QX ONE instrument. The thermocycling profile included a 45-minute MSRE incubation step prior to PCR amplification. Contrived samples were constructed by spiking DNA-free plasma with micrococcal nuclease-digested DNA from an amniotic fluid cell line ("fetal" component) and a B-lymphocyte cell line ("maternal" component). Clinical samples were remnant diagnostic samples with existing NGS non-invasive prenatal testing results attached. DNA was extracted from all samples with the Apostle MiniMax kit on the KingFisher Flex.

Results: From an initial set of 15 assays, a final five-assay multiplex was produced following amplicon sequencing with NGS and ddPCR screening. Although amplicon sequencing did not completely predict ddPCR performance and non- specific interactions, it was valuable for guiding the final ddPCR screen. The five-assay multiplex, consisting of three fetal assays and two maternal assays, produced an excellent linear response against contrived samples from 0% to 25% fetal fraction (R2 >0.99). Similarly, a high correlation was observed between ddPCR-estimated fetal fraction and NGS fetal fraction for a set of clinical samples (n=6, plus two non- pregnant controls, R2 >0.94).

Conclusions: As an epigenetic trait, DNA methylation is a useful way to discriminate between otherwise highly similar DNA sequences in an efficient and effective manner. Leveraging DNA methylation may be done with minimal impact to the standard ddPCR workflow. The high sensitivity, speed, and direct quantification of ddPCR make it an attractive alternative to NGS for fetal fraction estimation.