Fig 1.
Flowchart illustrating biomarker selection and validation.
Fig 2.
Levels of markers in whole blood, prostate normal (PNo) and cancer (PCa) tissues.
Methylation-specific multiplex ddPCR was performed on DNA extracted from whole blood, PNo and PCa tissue. Results are shown in box-plots as the ratio of detected copies/µl of each prostate DNA biomarker relative to the number of reference ALB molecules. (A) ACTRT2, (B) EVX1, (C) HOXD13, (D) DOCK2 and (E) HAPLN3. P-values were calculated using Mann-Whitney tests and are marked as follows: ns: p > 0.05; *: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001; ****: p ≤ 0.0001. WB: whole blood, PNo: normal prostate tissue, PCa: prostate cancer tissue.
Fig 3.
Limit of blank in plasma from control non-cancer patients.
Analysis of 4 ml plasma from men examined for prostate cancer, but with no histological findings of malignancy in the prostate. Samples were divided into a test cohort (N = 40) and a validation cohort (N = 40). The dot plots show the number of positive droplets found for the test cohort (left) and the validation cohort (right) for each assay in the multiplex, (A) ACTRT2, (B) EVX1, (C) HOXD13, (D) DOCK2, and (E) HAPLN3. The dashed line indicates the limit of blank (LOB) for each biomarker assay as determined in the test cohort.
Fig 4.
Levels of ACTRT2, EVX1, HOXD13, DOCK2, and HAPLN3 biomarkers in plasma from metastatic castration-resistant prostate cancer (mCRPC) patients.
(A) The log2 of the biomarker DNA copies +1 is shown for each biomarker in the multiplex. (B) The percentage of ctDNA detected. Blue = ACTRT2, green = EVX1, grey = HOXD13, Yellow = DOCK2, orange = HAPLN3.