Diagnostic Performance and Resource Utilization of Combining Blood Gene Expression, Cell-Free DNA, and Urine Chemokines for Monitoring Kidney Rejection

BACKGROUND: Following kidney transplantation, subclinical acute rejection (AR) occurs in 20-25% of clinically stable recipients; surveillance biopsies are invasive and often indicate no rejection. Moreover, the widespread use of noninvasive biomarkers is limited by cost. METHODS: We developed and sought to validate two-stage diagnostic models integrating urine chemokine assays (CXCL9 and CXCL10) with confirmatory blood-based gene expression profiling (GEP) and donor-derived cell-free DNA (dd-cfDNA). Data from two studies: 476 biopsy-paired samples from 226 recipients (discovery) and 144 samples from 134 recipients (validation), were analyzed retrospectively. Rapid, low-cost urine chemokine assays were used for stage-1 screening; recipients with elevated urine chemokines underwent additional stage-2 testing with GEP and/or dd-cfDNA. RESULTS: In the validation cohort, dd-cfDNA outperformed GEP for overall rejection and antibody-mediated rejection, whereas neither test validated significant discrimination for cellular rejection. CXCL9-based two-stage testing improved the area under the receiver operating characteristic curve (AUC) and positive predictive value (PPV) versus standalone stage-2 testing, while maintaining similar negative predictive value (NPV) and reducing stage-2 test use. At a probability screening threshold of 0.10, the CXCL9/Cr-based logistic regression (LR), adjusted for BK virus, urinary tract infection, sex, age, time post-transplant, and donor type, followed by dd-cfDNA validated a higher PPV (0.39 versus 0.34), lower sensitivity (0.56 versus 0.62), and similar NPV around 0.94, compared with dd-cfDNA alone, while recommending dd-cfDNA testing in only 0.79 (95% confidence interval, 0.72-0.85) of validation cases. Combined GEP and dd-cfDNA testing validated higher AUCs than dd-cfDNA as a second-stage test for overall (0.85 [0.79-0.94] vs. 0.82 [0.74-0.92]), antibody-mediated (0.96 [0.93-1.00] vs. 0.90 [0.79-1.00]), and cellular rejections (0.69 [0.61-0.89] vs. 0.63 [0.53-0.81]). CONCLUSION: This staged approach reduces unnecessary biopsies and guides the efficient use of costly blood-based biomarkers, while maintaining good clinical performance, particularly for subclinical antibody-mediated rejections.