Droplet digital PCR as a novel system for the detection of microRNA‑34b/c methylation in circulating DNA in malignant pleural mesothelioma

Our plain-English summary of Droplet digital PCR as a novel system for the detection of microRNA‑34b/c methylation in circulating DNA in malignant pleural mesothelioma, by H. Sato, J. Soh, K. Aoe, et al (International Journal of Oncology, 2019).

The study

• Authors: H. Sato, J. Soh, K. Aoe, N. Fujimoto, S. Tanaka, K. Namba, H. Torigoe, K. Shien, H. Yamamoto, S. Tomida, H. Tao, K. Okabe, T. Kishimoto, S. Toyooka
• Journal: International Journal of Oncology
• Published: 2019;54(6):2139-2148
• Cited by: 16 articles, as counted by NIH iCite
• Read it: PubMed · Publisher

Abstract

Malignant pleural mesothelioma (MPM) is a rare malignancy arising from the pleura that is difficult to diagnose, contributing to its dismal prognosis. Previously, we reported that the degree of microRNA (miR)‑34b/c methylation in circulating DNA is associated with the development of MPM. Herein, we present a newly developed droplet digital PCR (ddPCR)‑based assay for the detection of miR‑34b/c methylation in circulating DNA in patients with MPM. We originally prepared two probes within a short amplicon of 60 bp, designing one from the positive strand and the other from the complementary strand. The two probes functioned cooperatively, and our established assay detected DNA methylation accurately in the preliminary validation. We subsequently verified this assay using clinical samples. Serum samples from 35 cases of MPM, 29 cases of pleural plaque and 10 healthy volunteers were collected from 3 different institutions and used in this study. We divided the samples into 2 groups (group A, n=33; group B, n=41). A receiver‑operating characteristic curve analysis using the samples in group A determined the optimal cut‑off value for the diagnosis of MPM, with a sensitivity of 76.9% and a specificity of 90%. On the other hand, the use of the same criterion yielded a sensitivity of 59.1% and a specificity of 100% in group B, and corresponding values of 65.7 and 94.9% for the entire cohort, indicating a moderate sensitivity and a high specificity. In addition, when the analysis was focused on stage II or more advanced MPM, the sensitivity improved to 81.8%, suggesting the possibility that the methylated allele frequency in MPM may be associated with the stage of disease progression. On the whole, the findings of this study indicate that miR‑34b/c methylation in circulating DNA is a promising biomarker for the prediction of disease progression in patients with MPM.

In plain English

Researchers in Japan developed a blood test using droplet digital PCR to detect methylation (a chemical change) of the microRNA-34b/c gene region in DNA circulating in the blood. They tested it on stored serum from 35 people with malignant pleural mesothelioma, 29 people with pleural plaques and 10 healthy volunteers at three hospitals, collected between 2005 and 2015 before any treatment. Samples from one hospital (group A, 33 people) were used to set a cut-off for a positive result. With that cut-off, sensitivity was 76.9% and specificity 90% in group A, 59.1% and 100% in the other 41 samples (group B), and 65.7% and 94.9% across all 74 people. For stage II or more advanced mesothelioma, sensitivity was 81.8%. Positive droplet counts differed significantly among disease stages (P=0.02), with no significant difference among histological subtypes (P=0.16). The authors concluded that this methylation could be a promising biomarker for predicting disease progression. They noted a small sample, groups not matched for age and sex, and the use of serum rather than plasma. The article did not report participants' asbestos exposure.

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 development of a droplet digital PCR methylation assay, then a diagnostic accuracy test of it on stored serum samples from patients and controls
• Who was studied: Serum from 35 people with malignant pleural mesothelioma, 29 with pleural plaque and 10 healthy volunteers, collected January 2005 to January 2015 at Okayama University Hospital, Okayama Rosai Hospital and the National Hospital Organization Yamaguchi Ube Medical Center, Japan. Samples were split by collection site into group A (n=33, Okayama Rosai Hospital) and group B (n=41, the other two institutions). Blood was taken before any treatment.
• Exposure: The article does not report asbestos exposure for any participant. The factor measured was methylation of the miR-34b/c promoter region in DNA circulating in serum.
• Main results:
  • In group A, the optimal cut-off was at least 3 droplets above a fluorescence threshold of 7,000. This gave a sensitivity of 76.9% (10 of 13 mesothelioma cases positive) and a specificity of 90% (18 of 20 pleural plaque cases negative).
  • Using the same cut-off, group B had a sensitivity of 59.1% (13 of 22 mesothelioma cases) and a specificity of 100% (19 of 19 pleural plaque or healthy volunteer samples negative). The entire cohort had a sensitivity of 65.7% (23 of 35) and a specificity of 94.9% (37 of 39).
  • Restricted to stage II or more advanced mesothelioma, sensitivity was 81.8% (18 of 22 cases), with a specificity of 94.9%.
  • The number of droplets at or above 7,000 differed significantly among stages (one-way ANOVA, P=0.02). In pairwise comparisons, only stage I versus stage III was significant (P=0.03).
  • Methylation was not detected in one patient with clinical stage IV mesothelioma.
  • There was no significant difference in methylated allele frequency among the epithelioid, biphasic and sarcomatoid subtypes (P=0.16).
• The authors' conclusion: The authors concluded that miR-34b/c methylation in circulating tumor DNA could be a promising circulating biomarker for predicting disease progression in malignant pleural mesothelioma.
• Limitations the authors state:
  • The sample size was too small for a definitive conclusion, and the groups were not matched for background characteristics such as age and sex.
  • Serum was used, although plasma is more suitable for this kind of DNA analysis. The authors stated the assay should be validated using plasma samples.

Topics

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

Medical terms in this study, explained in our Lexicon: Pleural Mesothelioma, Pleura, Prognosis, Pleural Plaques, Disease Progression, Biomarker.

Related studies

• The degree of microRNA-34b/c methylation in serum-circulating DNA is associated with malignant pleural mesothelioma — T. Muraoka, J. Soh, et al. (2013)
• Are circulating microRNAs suitable for the early detection of malignant mesothelioma? Results from a nested case-control study — D.G. Weber, A. Brik, et al. (2019)
• 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)
• Circulating epigenetic biomarkers in lung malignancies: From early diagnosis to therapy — M. Tomasetti, M. Amati, et al. (2017)
• 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)
• The analysis of novel microRNA mimic sequences in cancer cells reveals lack of specificity in stem-loop RT-qPCR-based microRNA detection — P. Winata, M. Williams, et al. (2017)

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