Orthogonal Comparison of Molecular Signatures of Kidney Transplants With Subclinical and Clinical Acute Rejection: Equivalent Performance Is Agnostic to Both Technology and Platform.

We performed orthogonal technology comparisons of concurrent peripheral blood and biopsy tissue samples from 69 kidney transplant recipients who underwent comprehensive algorithm-driven clinical phenotyping. The sample cohort included patients with normal protocol biopsies and stable transplant (sTx) function (n = 25), subclinical acute rejection (subAR, n = 23), and clinical acute rejection (cAR, n = 21). Comparisons between microarray and RNA sequencing (RNA-seq) signatures were performed and demonstrated a strong correlation between the blood and tissue compartments for both technology platforms. A number of shared differentially expressed genes and pathways between subAR and cAR in both platforms strongly suggest that these two clinical phenotypes form a continuum of alloimmune activation. SubAR is associated with fewer or less expressed genes than cAR in blood, whereas in biopsy tissues, this clinical phenotype demonstrates a more robust molecular signature for both platforms. The discovery work done in this study confirms a clear ability to detect gene expression profiles for sTx, subAR, and cAR in both blood and biopsy tissue, yielding equivalent predictive performance that is agnostic to both technology and platform. Our data also provide strong biological insights into the molecular mechanisms underlying these signatures, underscoring their logistical potential as molecular diagnostics to improve clinical outcomes following kidney transplantation.

Molecular classifiers for acute kidney transplant rejection in peripheral blood by whole genome gene expression profiling.

There are no minimally invasive diagnostic metrics for acute kidney transplant rejection (AR), especially in the setting of the common confounding diagnosis, acute dysfunction with no rejection (ADNR). Thus, though kidney transplant biopsies remain the gold standard, they are invasive, have substantial risks, sampling error issues and significant costs and are not suitable for serial monitoring. Global gene expression profiles of 148 peripheral blood samples from transplant patients with excellent function and normal histology (TX; n = 46), AR (n = 63) and ADNR (n = 39), from two independent cohorts were analyzed with DNA microarrays. We applied a new normalization tool, frozen robust multi-array analysis, particularly suitable for clinical diagnostics, multiple prediction tools to discover, refine and validate robust molecular classifiers and we tested a novel one-by-one analysis strategy to model the real clinical application of this test. Multiple three-way classifier tools identified 200 highest value probesets with sensitivity, specificity, positive predictive value, negative predictive value and area under the curve for the validation cohort ranging from 82% to 100%, 76% to 95%, 76% to 95%, 79% to 100%, 84% to 100% and 0.817 to 0.968, respectively. We conclude that peripheral blood gene expression profiling can be used as a minimally invasive tool to accurately reveal TX, AR and ADNR in the setting of acute kidney transplant dysfunction.

Application of TruGraf v1: A Novel Molecular Biomarker for Managing Kidney Transplant Recipients With Stable Renal Function.

TruGraf v1 is a laboratory-developed DNA microarray-based gene expression blood test to enable proactive noninvasive serial assessment of kidney transplant recipients with stable renal function. It has been previously validated in patients identified as Transplant eXcellence (TX: stable serum creatinine, normal biopsy results, indicative of immune quiescence), and not-TX (renal dysfunction and/or rejection on biopsy results). TruGraf v1 is intended for use in subjects with stable renal function to measure the immune status as an alternative to invasive, expensive, and risky surveillance biopsies. MATERIALS AND METHODS: In this study, simultaneous blood tests and clinical assessments were performed in 192 patients from 7 transplant centers to evaluate TruGraf v1. The molecular testing laboratory was blinded to renal function and biopsy results. RESULTS: Overall, TruGraf v1 accuracy (concordance between TruGraf v1 result and clinical and/or histologic assessment) was 74% (142/192), and a result of TX was accurate in 116 of 125 (93%). The negative predictive value for TruGraf v1 was 90%, with a sensitivity 74% and specificity of 73%. Results did not significantly differ in patients with a biopsy-confirmed diagnosis vs those without a biopsy. CONCLUSIONS: TruGraf v1 can potentially support a clinical decision enabling unnecessary surveillance biopsies with high confidence, making it an invaluable addition to the transplant physician's tool kit for managing patients. TruGraf v1 testing can potentially avoid painful and risky invasive biopsies, reduce health care costs, and enable frequent assessment of patients with stable renal function to confirm the presence of immune quiescence in the peripheral blood.