Medical DNA technology for evaluating the sensitivity of luminal B breast tumors to neoadjuvant anthracycline chemotherapy based on DNA methylation markers

Objective. To develop a DNA technology for the prognosis of the effectiveness of neoadjuvant chemotherapy of breast cancer (BC) based on the determination of the methylation state of a limited set of DNA methylation markers by the method of multilocus methylation sensitive restriction enzyme polymerase chain reaction (MSRE-PCR). Material and methods. The research workflow was as follows. (1) Carry out a genome wide DNA methylation analysis on biopsy samples of breast tumors prior to anthracycline neoadjuvant chemotherapy. (2) Develop a limited panel of DNA methylation markers, the most informative for the prognosis of the effectiveness of neoadjuvant chemotherapy. (3) Develop and optimize the laboratory protocol of multi-locus MSRE-PCR for determining the methylation status of the markers in the panel. (4) Evaluate the diagnostic properties of the resulting panel of DNA methylation markers. Genome wide DNA methylation analysis of 27 BC biopsy specimens of the luminal B subtype, taken before the treatment under ultrasound guidance, was performed using the XmaI-RRBS method. According to the results of XmaI-RRBS, 10 genes have been selected, the state of methylation of the promoters of which most effectively marks epigenetic subtypes of tumors with different responses to neoadjuvant chemotherapy. Methylation status of the selected markers was next determined by MSRE-PCR with three primer pools in a sample of 40 BC biopsy specimens of the luminal B subtype taken before the treatment. Evaluation of the diagnostic properties of the system was carried out by ROC analysis. Results. By the genome wide DNA methylation analysis, the SLC9A3 , C1QL2 , DPYS , IRF4 , ADCY8 , KCNQ2 , TERT , SYNDIG1 , SKOR2 and GRIK1 gene regions were identified as the most informative markers of BC sensitivity to anthracycline neoadjuvant chemotherapy. To conduct locus-specific testing of the methylation status of these markers we have developed a multi-locus MSRE-PCR system. Based on the results of the locus-specific testing, the diagnostic properties of the system were determined: the area under the ROC curve was 84%, the sensitivity of the system was 82% with the specificity of 80%, the accuracy was 82%. Conclusion. The system including a limited number of DNA methylation markers, makes it possible to effectively predict the response of a luminal B subtype breast tumors to anthracycline neoadjuvant chemotherapy by an analysis of biopsy material obtained prior to treatment.

рак молочной железы, неоадъювантная химиотерапия, широкогеномный анализ метилирования ДНК, многолокусная метилчувствительная ПЦР, Breast cancer, neoadjuvant chemotherapy, genome wide analysis of DNA methylation, multilocus methylation sensitive restriction enzyme PCR

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