*Purpose: Non-invasive biomarkers distinguishing early signs of immune activation vs. quiescence could more objectively guide immunosuppression management after liver transplantation (LT). Our aim was to validate blood-based gene expression profiles that can serially detect immune activation prior to acute rejection (AR) distinct from immune quiescence (non-AR).

*Methods: Gene expression results in LT recipients with AR vs. non-AR (combination of other causes of graft dysfunction and normal graft function) were analyzed from two published cohorts (Northwestern University (NU); multicenter NIAID CTOT-14). A 70:30 approach (61 AR; 162 non-AR) was used for discovery and validation. Serial sample results before AR and non-AR were available in the CTOT-14 study (all <1 year post-LT). Microarray data was normalized utilizing a liver-specific fRMA vector. Variable selection was done using a specific variance (0.5), expression (8.0) and bootstrap (0.3) threshold.

*Results: Random forest modeling was used to generate a classifier on the discovery set distinguishing AR vs. non-AR (AUC 0.83; accuracy of 0.78, sensitivity 0.70, specificity 0.81, PPV 0.54, NPV 0.89; F-score .61). The final 59 probe model and locked probability threshold performed well on the validation set (accuracy of 0.72, sensitivity 0.67, specificity 0.73, PPV 0.48, NPV 0.86; F-score 0.56). The probability score line slopes were positive preceding AR and negative preceding non-AR and following AR treatment (all p<0.001; Figs. 1 & 2) in CTOT-14; the threshold was crossed at ~120 days prior to AR. Among the top 36 pathways that were significant using IPA (p <0.01) for the 59 probes, >40% were within immune response pathways, including allograft rejection.

*Conclusions: We have developed a blood-based biologically relevant genomic biomarker diagnostic for AR that can be detected prior to AR-associated graft injury and distinct from non-AR. Prospective interventional trials are needed to evaluate its utility in precision-guided immunosuppression following LT.

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