Clinical Validation of an Immune Quiescence Gene Expression Signature in Kidney Transplantation

Abstract

Background: Despite advances in immune suppression, kidney allograft rejection and other injuries remain a significant clinical concern, particularly with regards to long-term allograft survival. Evaluation of immune activity can provide information about rejection status and help guide interventions to extend allograft life. Here we describe the validation of a blood gene expression classifier developed to differentiate immune quiescence from both T cell mediated rejection (TCMR) and antibody-mediated rejection (ABMR). Methods: A five-gene classifier (DCAF12, MARCH8, FLT3, IL1R2, and PDCD1) was developed on 56 peripheral blood samples and validated on two sample sets independent of the training cohort. The primary validation set comprised 98 quiescence samples and 18 rejection samples: 7 TCMR, 10 ABMR, and 1 mixed rejection. The second validation set included 8 quiescence and 11 rejections: 7 TCMR, 2 ABMR, and 2 mixed. AlloSure donor derived cell-free DNA was also evaluated. Results: AlloMap Kidney classifier scores in the primary validation set differed significantly between quiescence (median 9.49, IQR 7.68-11.53) and rejection (median 13.09, IQR 11.25-15.28), p < 0.001. In the second validation set, the cohorts were statistically different (p = 0.028) and the medians were similar to the primary validation set. The AUC for discriminating rejection from quiescence was 0.786 for the primary validation and 0.800 for the second validation. AlloMap Kidney results were not significantly correlated with AlloSure, although both were elevated in rejection. The ability to discriminate rejection from quiescence was improved when AlloSure and AlloMap Kidney were used together (AUC 0.894). Conclusion: Validation of AlloMap Kidney demonstrated the ability to differentiate between rejection and immune quiescence using a range of scores. The diagnostic performance suggests that assessment of the mechanisms of immunological activity is complementary to allograft injury information derived from AlloSure dd-cfDNA. Together, these biomarkers offer a more comprehensive assessment of allograft health and immune quiescence.

Non-Invasive Monitoring for Rejection in Kidney Transplant Recipients After SARS-CoV-2 mRNA Vaccination

“…However, ddcfDNA levels may be less sensitive for the detection of T-cell mediated rejection (22), which is likely the reason it was not elevated in the patient who developed cellular rejection. The PBGEP (AlloMAP-Kidney) score derived from the expression of five candidate genes has been recently described as a marker of immune quiescence in KTRs (24). For discrimination between rejection and immune quiescence, it has an area under receiver operating characteristic curve of 0.78 and 0.89 as a standalone test and when combined with ddcfDNA, respectively (24).…”

“…PBGEP assay was performed and PBGEP score calculated as per AlloMAP protocol (CareDx, Inc., Brisbane, CA) (24). As described previously (24), peripheral blood was collected in PAXgene ® RNA tubes then shipped to CareDx, Inc. laboratories (Brisbane, CA) where RNA was extracted using the QIASymphony PAXgene Blood RNA kit (Qiagen, Cat no. 762635) following manufacturer's instructions.…”

“…Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) was run in triplicates for purified RNA samples and the mean C T for each determined as described previously (25). The mean C T for the five candidate genes (DCAF12, MARCH8, FLT3, IL1R2, and PDCD1) derived from previous studies in heart transplantation (26), was normalized to six reference genes (24). A multivariate model that integrates reference-normalized expression of the five genes computes a PBGEP (AlloMAP-Kidney) score (range 0-20) to differentiate immune quiescence from rejection with higher scores associated with rejection.…”

“…A multivariate model that integrates reference-normalized expression of the five genes computes a PBGEP (AlloMAP-Kidney) score (range 0-20) to differentiate immune quiescence from rejection with higher scores associated with rejection. Median (IQR) scores of 12.43 (11.12-14.29) and 10.19 (7.64-12.09) were seen in patients with and without kidney allograft rejection respectively (24). Fifty-one out of fifty-eight patients had PAXgene ® RNA tubes collected for PBGEP scoring.…”

“…To monitor for rejection non-invasively, we measured serum creatinine, UPCR, de novo DSAs by single-antigen bead assay, ddcfDNA as a marker of graft injury (22), 5-gene rejection PBGEP (24), and urinary CXCL9 mRNA levels using a CRISPR-Cas13 platform (27). No patients developed de novo DSAs or a significant increase in pre-vaccination DSA mean fluorescent intensities (MFIs) at three months post-vaccination (Figure 2A).…”