Development of a quantitative methylation-specific droplet digital PCR assay for detecting Dickkopf-related protein 3

Our plain-English summary of Development of a quantitative methylation-specific droplet digital PCR assay for detecting Dickkopf-related protein 3, by K. Araki, A. Kurosawa, H. Kumon (BMC Research Notes, 2022).

The study

• Authors: K. Araki, A. Kurosawa, H. Kumon
• Journal: BMC Research Notes
• Published: 2022;15(1):169
• Cited by: 6 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Objective: The detection and monitoring of DNA methylation status in circulating tumor cell DNA (ctDNA) provides critical insights into cancer diagnosis and progression. The methylation status of the Dickkopf-related protein 3 (DKK3) promoter region is correlated with the metastasis and recurrence of multiple cancers. Thus, detecting the methylation status via non-invasive methods is essential for the diagnosis and prognosis of cancers. Using a droplet digital polymerase chain reaction approach, we have developed a highly sensitive and quantitative measurement of methylated and unmethylated DKK3 derived from circulating cell-free DNA (ccfDNA).

Results: We confirmed the specificity of droplet digital methylation specific polymerase chain reaction (ddMSP). We selected the optimal bisulfite conversion method using commercially available kits. We validated the ddMSP analysis system by analyzing the methylation status of genomic DNA extracted from cultured mesothelioma cells and mesothelial cells. Our system quantified approximately 30 copies of cell-free DNA per 4 mL, which is sufficient for detecting ctDNA. Finally, we quantified methylated and unmethylated DKK3 copies in ccfDNA from 21 patients with malignant mesothelioma.

© 2022. The Author(s).

In plain English

This laboratory study developed a blood-test method, a methylation-specific droplet digital PCR assay, to count copies of the DKK3 gene's promoter region that carry or lack chemical tags called methylation, in cell-free DNA from blood. The article does not study or mention asbestos exposure. The authors reported that the test separated methylated from unmethylated DNA without non-specific reactions. Of three commercial bisulfite kits, they chose the EpiJET kit, which recovered approximately 50%. In cultured cells, methylated DKK3 was detected in the NCI-H28 and NCI-H226 mesothelioma cell lines, and to a lesser extent in NCI-H2052. It was not detected in MSTO-211H, NCI-H2452, the normal mesothelial cell line Met5A, or normal serum and plasma. The system quantified approximately 30 copies of cell-free DNA per 4 mL. In serum from 21 patients with malignant mesothelioma, 5–500 methylated copies per 4 mL were detected in eight patients and none in 13. Copies tended to increase with stage. The authors concluded the method selectively and quantitatively measured methylated and unmethylated DKK3. They noted they did not compare results with tumor samples.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: laboratory assay development study (research note) using cell lines, normal donor serum/plasma and patient serum
• Who was studied: Serum cell-free DNA from 21 patients with malignant mesothelioma (samples from clinical trials JapicCTI-152998 and JapicCTI-184040); also cultured mesothelioma cell lines, the mesothelial cell line Met5A, and normal human serum and plasma from donors; dates not given
• Main results:
  • Specificity tests: no non-specific reactions with the mismatched template in either assay
  • Among three bisulfite kits, the EpiJET kit showed a positive correlation with input, with a recovery rate of approximately 50%, and was selected
  • Cultured mesothelioma cells: methylated DKK3 was detected in NCI-H28 and NCI-H226, and to a lesser extent in NCI-H2052. It was not detected in MSTO-211H and NCI-H2452
  • No methylated copies were detected in the normal mesothelial cell line Met5A
  • The detection limit was approximately 30 copies of cell-free DNA per 4 mL of serum
  • The EpiJET kit had a coefficient of variation of 0.18, compared with 0.58 for the EZ DNA Methylation-Lightning Kit. The assay detected less than 0.2% methylated DNA
  • Malignant mesothelioma patients: 5–500 copies of methylated DKK3 per 4 mL of serum in eight patients, and no copies in 13 patients
  • The number of methylated copies tended to increase with stage. Differences by disease type could not be assessed because of the small number of cases
• The authors' conclusion: The authors concluded that their method selectively and quantitatively measured methylated and unmethylated DKK3 in circulating cell-free DNA, and that the assay can be used to analyze clinical samples
• Limitations the authors state:
  • Blood results were not compared with the patients' tumor samples, so it is unknown how well blood results reflect methylation in the tumor
  • Too few cases to discuss differences by disease type

Topics

The National Library of Medicine indexes this study under subjects our site covers: Mesothelioma.

Medical terms in this study, explained in our Lexicon: ctDNA, Metastasis, Prognosis, Mesothelioma, Mesothelial Cells.

Related studies

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• Tumor-specific genetic variants can be detected in circulating cell-free DNA of malignant pleural mesothelioma patients — M. Hylebos, K. Op de Beeck, et al. (2018)
• Treatment Response Monitoring in Patients with Advanced Malignancies Using Cell-Free SHOX2 and SEPT9 DNA Methylation in Blood: An Observational Prospective Study — L. de Vos, M. Jung, et al. (2020)
• Digital PCR for Detecting Loss of BAP1 and MTAP in Pleural Fluids of Mesothelioma Patients: Perspectives in the Diagnosis — G. Rosini, G. Alì, et al. (2026)
• DNA methylation status of REIC/Dkk-3 gene in human malignancies — T. Hayashi, H. Asano, et al. (2012)
• CDKN2A and MTAP Are Useful Biomarkers Detectable by Droplet Digital PCR in Malignant Pleural Mesothelioma: A Potential Alternative Method in Diagnosis Compared to Fluorescence In Situ Hybridisation — Y.Y. Cheng, M.L. Yuen, et al. (2020)

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