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. 2015 Oct 30;25(11):1250–1264. doi: 10.1038/cr.2015.126

Figure 4.

Figure 4

Identification of novel HCC-detecting markers in blood. (A) Volcano plot showing CGIs in plasma that are differentially methylated between the HCC patients with small tumors (n = 9, tumor size ≤ 3 cm) and cancer-free individuals (n = 45). The x axis shows the fold changes of the average methylation between the HCC patients and the cancer-free individuals, and the y axis shows the q-value as the FDR analogue of the P value (−Log10(q-value)) for a two-tailed MWW test of differences between two groups. The dashed line indicates statistical significance (FDR < 0.05). A total of 382 differentially methylated plasma CGIs (pdmCGIs) are identified (pink). (B) Venn plot view of the HCC-detecting markers (n = 41) and tdmCGIs (n = 866). (A, B) The type I and type II high-performance HCC-detecting markers are indicated in red and purple, respectively. (C) A heat map showing methylation of the HCC-detecting markers in the tissue samples. Each column represents a tissue sample of HCCs (T(HCC), red), adjacent livers (T(Aj), green), normal livers (T(NL), blue), WBCs (two biological replicates, yellow) or 10% FMG diluted in WBCs (brown), and each row represents a marker. The type I markers (n = 20) are ranked by their methylation levels in the liver calculated by their MePM values in the normal liver relative to the 10% FMG diluted in WBCs from the lowest to the highest. The type II markers (n = 21) are clustered using the hierarchical clustering. In the heat map, blue color indicates low, white and yellow intermediate and red high DNA methylation values, shown by log2(MePM). The markers for the classifiers are boxed. (D) Boxplots of the representative markers of the type I (ST8SIA6) and the type II [SHANK2(i)] HCC-detecting markers showing their methylation in plasma (red) or tissues (blue) samples. **P < 0.01; ND, no statistical difference. Two-tailed MWW test. (E) Scatter plots of the methylation levels of WBCs vs the normal liver. The RRBS results of 3 886 CGI loci are shown. Red circles indicate the type I markers (n = 19); purple circles indicate the type II markers (n = 20), and solid circles indicate the markers for the classifiers.