Detecting plasma SHOX2, HOXA9, SEPTIN9, and RASSF1A methylation and circulating cancer cells for cholangiocarcinoma clinical diagnosis and monitoring

Figure 1 Analysis of SHOX2, RASSF1A, SEPTIN9, and HOXA9 gene expression in normal tissue (n = 9) and cholangiocarcinoma tissue (n = 36) from previously reported genome data. A-D: SHOX2, RASSF1A, SEPTIN9 and HOXA9 gene expression was upregulated in cholangiocarcinoma (CCA) tissue (n = 36); E-H: Beta values ranging from 0 (unmethylated) to 1 (fully methylated) indicate the DNA methylation levels of the SHOX2, RASSF1A, SEPTIN9 and HOXA9 promoters in normal tissue (n = 9) and CCA tissue (n = 36). CCA: Cholangiocarcinoma.

Figure 2 Quantitative analysis of SHOX2, RASSF1A, SEPTIN9, and HOXA9 DNA methylation in cholangiocarcinoma (n = 156) and control (n = 98) specimens. CCA: Cholangiocarcinoma.

Figure 3 The receiver operating characteristic curves, calculated area under the curves, and sensitivity and specificity values of the four DNA methylation biomarkers in the plasma of patients with cholangiocarcinoma and the receiver operating characteristic curves for circulating tumor cells to assess cholangiocarcinoma progression. A: The receiver operating characteristic (ROC) of SHOX2; B: ROC of HOXA9; C: ROC of RASSF1A; D: ROC of SEPTIN9; E: ROC of circulating tumor cell for progression. The ROC curves and resulting area under the curve (AUC) for 96 patients with cholangiocarcinoma are depicted. AUC-SHOX2 = 0.799 (95%CI: 0.724-0.874), AUC-SEPTIN9 = 0.713 (95%CI: 0.632-0.795), AUC-RASSF1A = 0.765 (95%CI: 0.689-0.841), AUC-HOXA9 = 0.777 (95%CI: 0.705-0.850). AUC-circulating tumor cells = 0.653 (95%CI: 0.533-0.774). ROC: The receiver operating characteristic; CTC: Circulating tumor cell.

Figure 4 The receiver operating characteristic curves and resulting area under the curve values for patients with cholangiocarcinoma (n = 156) and early cholangiocarcinoma (n = 11). A: The receiver operating characteristic curves of complex methylation and carbohydrate antigen 19-9 (CA199) of the patients. area under the curve (AUC)-complex methylation = 0.828 (95%CI: 0.764-0.891), AUC-CA199 = 0.746 (95%CI: 0.666-0.826), AUC-complex methylation + CA199 = 0.851 (95%CI: 0.792-0.910); B and C: The AUC of the complex evaluation and CA199 for diagnosing early cholangiocarcinoma. AUC-complex methylation = 0.682 (95%CI: 0.654-0.710), AUC-CA199 = 0.542 (95%CI: 0.358-0.726). CA199: Carbohydrate antigen 19-9.

Figure 5 The relapse-free survival and overall survival curves for the initial circulating tumor cell counts of the patients with cholangiocarcinoma, and the relapse-free survival curve for RASSF1A methylation. A and B: The progression free survival and overall survival curves for the initial circulating tumor cell counts before therapy in the patients with cholangiocarcinoma; C: The progression free survival curves for the RASSF1A methylation in the patients with cholangiocarcinoma. CTC: Circulating tumor cell.

Figure 6 Kaplan-Meier curves modeling the effect of methylation on overall survival in cholangiocarcinoma. Overall survival was calculated from plasma sampling time until death from any cause, and cases were censored at last follow-up. The effect of high and low methylation levels of SHOX2, RASSF1A, SEPTIN9, and HOXA9 were evaluated using a univariate approach with the log rank method. A: SHOX2; B: RASSF1A; C: SEPTIN9; D: HOXA9. Survival curves were estimated with the Kaplan-Meier method. Group definition: The cholangiocarcinoma samples were divided into high (DCtSHOX2 ≤ 9; DCtRASSF1A ≤ 12; DCtSEPTIN9 ≤ 9; DCtHOXA9 ≤ 8) and no/low methylation levels (DCtSHOX2 > 9; DCtRASSF1A > 12; DCtSEPTIN9 > 9; DCtHOXA9 > 8). HR: Hazard ratio.

Figure 7 Nomograms to predict the diagnosis of patients with cholangiocarcinoma. The nomograms comprise the levels of carbohydrate antigen 19-9, methylation, and other significant indicators. The points total is located on the total point axis, and a vertical line is traced down to the diagnostic axes to predict the likelihood of a risk. CTC: Circulating tumor cell; CA199: Carbohydrate antigen 19-9.

Figure 8 Internal cross-validation calibration curves at 1 and 2 years.