Abstract 4153: Usefulness of sensitive digital PCR assay to quantify microRNA-34b/c methylation in the circulating serum DNA of malignant mesothelioma patients
Authors: Takayuki Muraoka, Junichi Soh, Shinichi Toyooka, Nobukazu Fujimoto, Keisuke Aoe, Hiromasa Yamamoto, Kazuhiko Shien, Masashi Furukawa, Yuho Maki, Norimitsu Tanaka, Tsuyoshi Ueno, Hiroaki Asano, Kazunori Tsukuda, Takumi KIshimoto, Takemi Otsuki, Shinichiro Miyoshi
Journal: Cancer Research (2012)

Abstract . Background: Malignant mesothelioma (MM) is an aggressive tumor but it can sometimes be cured if its clinical stage is not advanced. It is very expected to establish a useful screening test for early detection of MM. Recently we have found that DNA methylation of microRNA-34bc (miR34b/c) plays a dismal role on pathogenesis of MM and is frequently observed in MM. Circulating DNA from malignant tumors is known to be found in the serum of the patient. Digital polymerase chain reaction (PCR) is a high sensitive quantitative assay using real time PCR assay. In order to establish a new early detection system for MM, we quantify the extent of DNA methylation of miR-34b/c using a digital PCR assay in the circulating serum DNA from MM. Material and methods: We collected serum samples from 30 MM patients, 22 patients with benign asbestos pleurisy (BAP), and 10 healthy volunteers (HV) at Okayama Rosai Hospital, NHO Yamaguchi Ube Medical Center and our institute during January 2006 to August 2009. The circulating serum DNA was extracted using QIAamp Circulating Nucleic Acid Kit (Qiagen) followed by bisulfite conversion using EpiTect Bisulfite Kits (Qiagen) according to manufactural protocols. The real time PCR assay of methylation-specific PCR (MSP) for miR-34b/c was performed using SYBR Green method. In addition, the digital PCR of real time MSP assay was explored for 40 wells per each sample with a suitable dilution to aim the increase of sensitivity of PCR reaction, and we quantified the extent of methylation in each sample by counting the number of positive well of PCR reaction per sample. As positive and negative controls, the supernatant cultured medium of NCI-H290 harboring heavy methylation of miR-34b/c and water blank was used, respectively. Results: By melting curve analysis, we distinguished the miR-34b/c methylated wells from unmethylated wells based on the temperatures of PCR products of them (78.2 degree on methylated samples vs. 76.2 degree on un-methylated samples). Using the criteria above, each 40 wells per sample was defined as positive or negative for miR-34b/c methylation. The miR-34b/c methylated samples were defined as having more than three positive wells by ROC curve analyses. Finally, we found that the number of positive wells was significantly higher in MMs than that in other groups, and the miR-34b/c methylated samples was observed in 87% of MM patients, 45% of the patients with BAP, and 10% of HV, respectively, indicating that the miR-34b/c methylation in MM patients were significantly frequent than that in others (P &amp;lt;0.0001). Conclusion: The digital PCR assay can detect miR-34b/c methylation in the circulation serum DNA of MM patients to find that miR-34b/c methylation is more frequent in MM than in BAP, suggesting that it may become a useful high-sensitive screening test to distinguish MM patients from the patients with BAP. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4153. doi:1538-7445.AM2012-4153