Introduction: There are no clinically established blood-based diagnostic biomarkers for urothelial carcinoma patients. Amino acids (AA) have two isoforms of L- and D-AA and D-AA have been believed to be physiologically nonexistent. However, it becomes clear that some D-AA has clinical significance in the human body. Here, we evaluated circulating D-AA levels in the blood and their clinical possibilities as diagnostic biomarkers in urothelial cancer patients. Methods: We enrolled 345 patients just before surgery for urothelial carcinoma (UC) (n=123), renal cell carcinoma (RCC) (n=143), and healthy donor of kidney transplantation (n=79). We collected 345 serum samples just before surgery and 16 samples one week after surgery. D-AA levels in serum were measured using two-dimensional high-performance liquid chromatography. We evaluated its usefulness as a diagnostic biomarker of UC in the derivation cohort (n=22) and two independent validation cohorts in 254 (Cohort-1) and 69 (Cohort-2) participants, respectively. Results: In the derivation cohort, only four D-AA (D-Asparagine (D-Asn), D-Serine (D-Ser), D-Alanine (D-Ala), and D-Proline (D-Pro)) could be measured in serum samples among 22 kinds of D-AA. D-Asn and D-Ala levels were significantly higher in UC patients compared to RCC patients and healthy donors (p < 0.05 and p<0.01, respectively). The area under the curve (AUC) of each D-Asn to diagnose UC was 0.800. Sensitivity and specificity were 100% and 60% using a cut-off level of 0.233 IU/ml. In validation Cohort-1, D-Asn levels had a high accuracy of diagnosis for UC (AUC: 0.851). Sensitivity and specificity were 77.8% and 79.3% using the same cut-off level as the derivation cohort. In validation cohort-2, D-Asn levels had the high accuracy of diagnosis for UC (AUC: 0.836) and its sensitivity and specificity were 61.9% and 81.2% using the same cut-off level as the derivation cohort. Also, serum D-Asn levels of 16 UC patients just after surgery significantly decreased compared to those just before surgery (p=0.005). Conclusions: D-Asn levels in the blood could become the promising diagnostic markers of UC patients. SOURCE OF Funding: This work was supported by a KAKENHI grant (22H03213).
