Abstract 70P
Background
Emergence of genome-wide DNA methylation screening techniques allowing identification of differentially methylated regions that discriminate groups of clinically different samples has stimulated development of epigenetic diagnostic tests, especially for applications in cancer diagnostics. Translation of markers identified on genome-wide datasets into clinically applicable tests requires specific test systems design and validation on independent cohorts. Objective of this study was to select DNA methylation markers for prediction of luminal B breast cancer (BC) neoadjuvant chemotherapy (NACT) response from the genome-wide DNA methylation sequencing results; to design quantitative PCR assays for their detection, and to estimate their diagnostic values in order to provide a panel for accurate prediction of response to NACT.
Methods
Genome-wide methylation analysis of 43 luminal B subtype BC biopsy specimens taken before NACT was performed by bisulfite sequencing (XmaI-RRBS). Informative DNA methylation markers were selected from XmaI-RRBS data using the Wilcoxon–Mann–Whitney test. For these markers, Methylation Sensitive Restriction Enzyme qPCR (MSRE-qPCR) assays were designed and an independent cohort of 35 luminal B samples was assessed. Most informative markers were combined in panels and assessed by multiplex MSRE-qPCR. The area under the ROC curve (AUC) with cross-validation was calculated for each panel.
Results
Best NACT response DNA methylation classifier for luminal B breast cancer includes CpG islands of TTC34, LTBR and CLEC14A genes. For LTBR, lower methylation levels predict higher NACT efficacy, with AUC of 69%, while for TTC34 and CLEC14A higher NACT efficacy was associated with higher methylation levels, with AUCs of 56% and 57% respectively. Combination of the three markers in a panel enhances diagnostic values to AUC of 76%, sensitivity of 70% and specificity of 79%.
Conclusions
A three-gene DNA methylation markers panel including TTC34, LTBR and CLEC14A genes accurately predicts luminal B breast cancer response to NACT and is adapted for potential clinical application, being implemented as a multiplex MSRE-qPCR assay for use with real-time PCR instruments.
Clinical trial identification
Editorial acknowledgement
Legal entity responsible for the study
Research Centre for Medical Genetics, Moscow, Russian Federation.
Funding
Russian Science Foundation (project No.18-15-00430).
Disclosure
All authors have declared no conflicts of interest.