Subsequently, a spiking experiment was performed to assess both sensitivity and reproducibility in dependence on a distinct number of RT112 tumor cells (range: 100–10,000 cells), i.e., RT112 bladder cancer cells harboring methylated ECRG4 (see Supplementary Figure S1) and ITIH5 [20] genes were spiked into 20 mL pooled urine of healthy donors (n = 4), respectively. The gene discussed is ECRG4; the disease is urinary bladder carcinoma.