Background: Sub-clinical acute rejection (SCAR) is histologic acute cellular rejection in the presence of a normal/stable serum creatinine, and is associated with worse graft survival. Though protocol biopsies (PBx) are performed at some centers to detect SCAR there is a pressing need for a reliable, non-invasive biomarker for SCAR. Such a test will reduce the need for PBx, inform the need for a “for cause” biopsy and help monitor and adjust therapy. We describe discovery and validation of a gene expression profile in the peripheral blood of patients with SCAR that is different from that of patients with normal PBx (TX – normal creatinine) and patients with clinical acute cellular rejection (CAR – elevated creatinine) found on a “for cause” biopsy.

Methods: A total of 124 samples from the CTOT08 trial (33 SCAR, 31 CAR and 60 TX) were profiled using Affymetrix HT 133 PM Plus Arrays. Clinical validation was done using 69 samples from the Northwestern repository (21 CAR, 23 SCAR, and 25 TX). We performed a 3-way ANOVA analysis of CAR vs. SCAR vs. TX. We used the Nearest Centroid (NC) algorithm to build predictive models and used Optimism Corrected Bootstrapping to adjust for over-fitting of data. We then took the signals from the classifier genes and transformed the raw gene expression results into a simple numerical score to create a tool for serial monitoring.

Results: From over 2,500 significantly differentially expressed probesets (FDR<10%) we derived a 61 (FDR<5%) probeset classifier to create a diagnostic signature. This signature gave optimism corrected AUCs ranging from 0.86-0.89. The 3-way classifier validated on the Northwestern samples, with AUCs ranging from 0.83-0.90. Our molecular scoring system revealed clear separations between each key phenotype and demonstrated an increase from TX to SCAR to CAR.

Conclusions: We have discovered and clinically validated a gene expression profile in the peripheral blood of patients with SCAR. This gene expression profile (SCAR) differs from a previously validated peripheral blood signature for patients with clinical acute cellular rejection found on a “for cause” biopsy. Our study shows the first blood based signature for SCAR. It also demonstrates the value of integrating predictive molecular biomarkers into clinical practice to serially monitor and improve long-term outcomes for kidney transplant patients.

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