Figure 3.

Biological and clinical significance of age-PCGT DNAm signature. (A,B) Average methylation values of the 69 age-hypermethylated and 20 age-hypomethylated PCGT CpGs as a function of disease status in 48 cervical cytology samples. (HPVneg) Normal cervical sample not infected with HPV, (HPVpos) normal cervical sample infected with HPV, (HPVpos-Dysplasia) samples infected with HPV and displaying dysplasia. Wilcoxon test P-value between normal and dysplastic condition is given. Number of samples in each group given below boxplots. (C) Histogram distribution of −log₁₀(P-values) from 1000 randomly selected 69 non-age-associated PCGT CpGs. P-values were derived from the Wilcoxon test. (Red line) −log₁₀(P-value) for the 69 age-hypermethylated PCGT CpGs, (blue line) −log₁₀(P-value) for PCGT CpGs not mapping to age-PCGTs. In less than 0.5% of runs (P < 0.01) were P-values as extreme as the observed one, indicating that the age-PCGTs discriminate the dysplastic condition better than a random set of PCGTs. (D) Heatmap of the 48 cervival samples over the 69 age-hypermethylated PCGT CpGs. Samples were clustered using a Gaussian mixture model and three optimal clusters were inferred using the Bayesian Information Criterion (see Supplemental material). (Orange, brown, pink) Distinct clusters. The disease status of samples is labeled as a color bar (PROGR): (light green) HPVneg, (green) HPVpos+normal, (red) HPVpos+dysplasia. CpGs were clustered according to hierarchical clustering with a Pearson correlation metric. Prior to sample and CpG clustering, methylation profiles of invividual CpGs were renormalized to mean zero and unit standard deviation. Heatmap reflects, for each CpG, relative methylation levels across samples as determined by the renormalized methylation profile. (Blue) Relative high methylation, (yellow) relative low methylation. (E,F) Average gene expression intensity (Affymetrix) values for the 64 age-hypermethylated PCGTs in normal ovarian (OvN) and ovarian cancer tissue (OvC) and in normal cervix (CVX-N) and cervical cancer (CVX-T). Number of samples of each type and Wilcoxon test P-values are given.