Survival analysis of the MESCs demethylation instability index in various cancers.

<p>Kaplan-Meier survival curves between the upper (blue) and lower (green) tertiles of the demethylation instability index (DeMI) at MESCs in (A) Endometrial cancer, (B) Ovarian cancer, (C) Cervical cancer, and (D–E) two Breast Cancer cohorts. The hazard ratio (HR), 95% confidence interval (CI) and P-values are from the multivariate Cox regression model, with “n” denoting the number of samples in cohort. Clinical endpoint used is indicated on the y-axis (OS = overall survival, RFS = relapse free survival).</p

Data sets used in this study.

<p>Cervical cells collected three years before half of the patients developed dysplasia (BDy), normal versus dysplastic cervical cells (Dy), normal cervical tissue and invasive cervical cancer (Cvx CA), non-neoplastic breast tissue and breast cancer (BC), normal endometrium and endometrial cancer (EC) and metastatic endometrial cancer (EC (Meta)), ovarian cancer tissue (OvC; The Cancer Genome Atlas, TCGA), colon cancer tissue (ColC), lung cancer tissue (LC) and normal and cancer associated lung fibroblasts (LC Fibro), and white blood cell (WBC) samples from <i>BRCA1</i> mutation carriers and controls (<i>BRCA1</i>). The number of samples, their distribution in terms of normal and cancer, cell-type, age-range (years) and reference to data access is given. GSE numbers are GEO accessions. NA, not available.</p

Heatmap of the top 50 most frequently hypomethylated MESCs in cancers.

<p>The CpGs show stable fully methylated states across all normal samples. Methylation values: blue = high methylation (β-value>0.7), skyblue = hemi methylation (0.25<β-value<0.7), yellow = low methylation (β-value<0.25). Sample labels at the top of the heatmaps: normal (grey) and cancer (black).</p

Differential dynamics of hypermethylated and hypomethylated PCGTs and MESCs.

<p>Bar charts representing percentages of significantly hypermethylated (blue) and hypomethylated (orange) PCGT and MESC CpGs in (A) each stage of cervical carcinogenesis: Cervix ‘Before Dysplasia’, ‘Dysplasia’, and ‘Invasive Cancer’, all relative to normal cervical cells or tissue; and in (B) ‘Breast CA’, ‘Endo CA’, ‘Colon CA’, and ‘Lung CA’, all relative to their respective normal controls. The significance of the binomial test assessing skew of hypermethylated versus hypomethylated is indicated by ‘*’, ‘**’, and ‘***’ for P-value<0.05, 0.01, and 0.001 respectively. (C) and (D) are the scatterplots of the age-adjusted linear regression t-statistics against their corresponding −log10(P-values) testing the association with the normal and lung cancer fibroblasts on the colon-PMD PCGTs and colon-PMD MESCs respectively.</p

Methylation changes between normal, primary, and metastatic endometrial cancer.

<p>(A) Boxplots comparing the frequency of PCGT (0→1/2) DNA methylation changes, and the frequency of combined MESC (1→0) and MESC (2→0/1) DNA methylation changes (“combined DeMI index”), between normal endometrium (N), primary endometrial cancer (C), and between normal endometrium and metastatic endometrial cancers (MET). One-tailed Wilcoxon rank sum test P-values for the instability indices between cancer and metastases are indicated. (B) Receiver operating characteristic (ROC) curves measuring the dissimilarity in the combined DeMI index between primary and metastatic endometrial cancers with corresponding Area Under Curve (AUC) and 95% CI.</p

Definition of epigenetic instability indices and dynamics of PCGT and MESC methylation in cancer.

<p>(A) Definition of epigenetic instability indices. Shown are the six possible DNA methylation changes between normal and cancer tissue. Thresholds used to define unmethylated (yellow), hemimethylated (skyblue) and fully methylated (blue) CpGs are described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002517#s4" target="_blank">Materials and Methods</a>. Stable MESC (or PCGT) CpGs are defined by MESC (or PCGT) CpGs, which have the same methylation state in all normal samples. The Epigenetic Instability Index (EpI) is then defined as the fraction of stable CpGs altered in cancer. We defined 4 separate indices to describe the transitions: 0→1/2, 1→2, 1→0, 2→1/0. The index describing alterations from a fully methylated to either a hemi or unmethylated state is called the Demethylation instability index (DeMI). (B) Dynamics of PCGT and MESC DNA methylation in cancer. Diagram illustrates the differential dynamics of PCGT and MESC CpG DNA methylation in cancer. Most PCGTs start out unmethylated (white lolly-pops) in normal cells but acquire DNA methylation (black lolly-pops) in normal cells 3 years before the emergence of dysplasia (BDy). PCGT hypermethylation increases further with Dysplasia (Dy) and cancer, but is not a strong determinant of invasion or poor outcome (metastasis). In contrast, most MESCs start out either fully or hemi methylated in normal cells, and gradually lose methylation during the progressive stages of cancer, with hypomethylation at MESCs a key determinant of metastases and poor outcome.</p

Anti-correlation between the TET mRNA expression level and methylation β-value.

<p>Heatmap of the 250 most (upper half) and least (lower half) anti-correlated hypomethylated CpGs in cervical cancer samples ranked according to their TET3 mRNA expression levels from the lowest (green) to the highest (red) with the indication of MESCs (purple) and nonMESCs (white). The odds ratio (OR) and P-value (P) are obtained from Fisher's exact test estimating the enrichment of MESCs among hypomethylated CpGs that are significantly anti-correlated with TET3 mRNA expression level.</p

Methylation profile of PCGTs and MESCs in cervix data.

<p>Scatter plots of mean β-values in normal cervical tissue (x-axes) vs. cervical cancer tissue (y-axes) of (A) all CpGs with PCGTs highlighted in brown and (B) all CpGs with MESCs highlighted in purple. (C) is the bar chart indicating the enrichment odds ratios (OR) and P-values (Fisher-test), testing for enrichment of PCGTs among CpGs unmethylated in normal cervix (mean β-value<0.2) and with a higher mean β-value in (i) normal samples which develop dysplasia (BDy), (ii) non-invasive dysplastic samples (Dy), and (iii) invasive cervical cancer (CA); (D) the bar chart indicating the enrichment of MESCs among those CpGs methylated in the normal cervix (mean β-value>0.4) and with a lower mean β-value in tissue representing the three stages of cervical carcinogenesis (BDy, Dy, and CA).</p

Survival analysis results of the PCGTs and MESCs Epigenetic Instability Index (EpI) in women's cancers.

<p>Univariate and multivariate Cox regression results for the PCGT EpI and MESC EpI (DeMI) in endometrial, ovarian, cervical and breast cancer with number of samples (n), Hazard ratio (HR), 95% confidence interval (CI), and P-value (P). Overall survival was used for endometrial, ovarian and breast cancer, relapse free survival for cervical cancer.</p