Figure 2.

DNA methylation-associated silencing of CDO1 is cancer specific, frequent, and correlates with disease progression and outcome. A, DNA methylation and protein expression status of CDO1. CDO1 promoter region from −168 bp to −45 bp relative to the TSS was assayed by MSP in a cohort of 20 normal breast tissues from non–cancer patients and 185 primary breast cancers (BC) of stages 0 (DCIS) to 4 with U and M marking unmethylated and methylated bands, respectively. Representative examples are shown for each cohort and each tumor stage. In vitro methylated DNA (IVD), DKO cells, normal lymphocytes (NL), and H₂O controls were assayed along with samples. Protein expression status of CDO1 was assayed by immunohistochemistry in normal breast and selected primary breast cancers. Note, BC013a, a DCIS sample, displays loss of CDO1 expression in hyperproliferative ductuli (HD) but expression of CDO1 in normal ductuli (ND). B, scatter plot depicting correlation between expression log₂ values (y-axis; analyzed on Agilent 244 K Custom Gene Expression G4502A-07 platform) and DNA methylation β values (x-axis; analyzed on Illumina HumanMethylation 27k platform) of CDO1 in 255 primary breast cancers from TCGA data portal. A Spearman correlation coefficient of ρ = −0.47 and P value of 1.7e-15 were calculated. A P < 0.05 was considered statistically significant. C, DNA methylation frequency (in%) of CDO1 plotted by tumor stage of 185 primary breast cancers. CDO1 methylation status and tumor stage were correlated using Pearson χ² test. ^*, P < 0.05. D, survival curve depicting prognostic value of CDO1 methylation status for outcome prediction in 185 primary breast cancers. HR for prognostic value was calculated using univariate Cox proportional hazards regression model.