Flow diagram of the study.

Development and validation of tissue diagnosis model based on the 27 CRC-specific DMRs. (A) The use of ROC curve analysis to assess the performance of the tissue diagnostic model in differentiating CRC patients from normal individuals of the training cohort (51 tissues we collected). (B-C) The use of ROC curve analysis to assess the performance of the tissue diagnostic model in differentiating CRC patients from normal individuals(B) and distinguish AA from Normal subjects (C) in tissue validation cohort 1(GSE 48684 dataset). (D-E) The sensitivity and specificity of the diagnosis model in the tissue training(D) and validation cohort 1(E).

Development and validation of a plasma diagnosis model based on the 27 CRC-specific DMRs. (A-B) Heatmap illustrating the DMRs between CRC and advanced adenoma and healthy controls in the training (A) and validation cohort (B). (C) The use of ROC curve analysis to assess the performance of the diagnosis model in differentiating CRC patients from normal individuals of the training cohort. (D) The use of ROC curve analysis to evaluate the performance of the diagnosis model in differentiating CRC patients from normal individuals of the validation cohort. (E-G) In the validation cohort, the performance of the diagnosis model in differentiating early-stage CRC (0-Ⅱ) from normal individuals (E); in differentiating advanced-stage CRC (Ⅲ-Ⅳ) from normal individuals (F); in differentiating AA from normal individuals (G). (H) Methylation scores of Normal AA and CRC individuals generated from the diagnosis model in the training and validation cohort (The dashed line represents the cutoff value). (I) The sensitivity and specificity of the diagnosis model stratified by stages in the training and validation cohort. Abbreviations: DMRs, differential methylated regions; ROC, receiver operating characteristic; AA, advanced adenoma; CRC, colorectal cancer.

External validation of plasma diagnosis model. (A-C) In the external validation cohort, the performance of the diagnosis model in differentiating early-stage CRC (0-II) from normal individuals (A); in differentiating AA from normal individuals (B); in differentiating NAA from normal individuals (C). (D) Methylation scores of Normal, NAA, AA and CRC (0-II) individuals generated from the diagnosis model in the external validation cohort (The dashed line represents the cutoff value). (E) The sensitivity and specificity of the diagnostic model for NAA, AA, and CRC in the external validation cohort.

Development and validation of a plasma-based metastasis model and a plasma-based prognosis model. (A-B) The methylation scores generated by the diagnosis model for CRC patients across various M stages (A) and different disease stages (B). (C) ROC curve analysis was performed to assess the performance of 27 DMRs methylation scores generated by the diagnosis model in distinguishing M1-CRC patients from M0-CRC patients in the training (AUC = 0.969) and validation (AUC = 0.955) cohorts. (D) ROC analysis evaluates the performance of 27 DMRs methylation scores generated by the diagnosis model in predicting the prognosis of CRC patients. (E) Kaplan-Meier survival curves comparing OS between the high-risk CRC group and low-risk CRC group of prognosis model in the training cohort and validation cohort. (F, H) Metastasis and prognosis prediction of the training and validation patitients(n = 88). The black dotted line at the cutoff value divides the patients into high-risk and low-risk groups. Yellow circles and gray circles represent patients without distant metastasis and with distant metastasis, respectively (F). Yellow circles and gray circles represent patients survived and deceased, respectively (H). (G, I) The proportion of metastasis and deceased is higher in the high-risk group. The p-value was calculated using a two-sided Fisher’s exact test. Abbreviations: DMRs, differential methylated regions; ROC, receiver operating characteristic; M1-CRC, colorectal cancer with distant metastasis; M0-CRC, colorectal cancer without distant metastasis; AUC, the area under the curve.

Methylation scores of paired tissue and plasma samples. (A) The methylation scores of tissues from Normal, AA, CRC 0-Ⅱ, CRC Ⅲ-Ⅳ individuals. (B) The methylation scores of blood samples from Normal, AA, CRC 0-Ⅱ, CRC Ⅲ-Ⅳ individuals. (C) The mechanism diagram interpreting the methylation scores changes of tissue and plasma across different populations.