A Peripheral Blood Gene Expression Signature to Diagnose Subclinical Acute Rejection
Authors: Zhang Weijia , Yi Zhengzi , Keung Karen L. , Shang Huimin , Wei Chengguo , Cravedi Paolo , Sun Zeguo , Xi Caixia , Woytovich Christopher , Farouk Samira , Huang Weiqing , Banu Khadija , Gallon Lorenzo , Magee Ciara N. , Najafian Nader , Samaniego Milagros , Djamali Arjang , Alexander Stephen I. , Rosales Ivy A. , Smith Rex Neal , Xiang Jenny , Lerut Evelyne , Kuypers Dirk , Naesens Maarten , O’Connell Philip J. , Colvin Robert , Menon Madhav C. , Murphy Barbara 
Published: July 2019

Significance Statement
Biomarkers for noninvasive diagnosis of subclinical acute rejection are needed to enable risk-stratification and tailoring of immunosuppression for kidney transplant recipients. Using RNA sequencing analyses of whole blood collected from a cohort of transplant recipients at the time of surveillance biopsy, the authors identified a transcriptional signature on the basis of a set of 17 genes that accurately detects ongoing subclinical rejection. After extensive validation, they developed a sequencing-based targeted expression assay on the basis of this gene set that was able to identify subclinical rejection at 3 months post-transplant and increased risk of graft loss in an independent cohort of 110 patients. This assay represents a potentially useful tool to monitor kidney transplant recipients and optimize immunosuppressive therapy, although larger studies are needed to validate the assay’s clinical utility.

Background
In kidney transplant recipients, surveillance biopsies can reveal, despite stable graft function, histologic features of acute rejection and borderline changes that are associated with undesirable graft outcomes. Noninvasive biomarkers of subclinical acute rejection are needed to avoid the risks and costs associated with repeated biopsies.

Methods
We examined subclinical histologic and functional changes in kidney transplant recipients from the prospective Genomics of Chronic Allograft Rejection (GoCAR) study who underwent surveillance biopsies over 2 years, identifying those with subclinical or borderline acute cellular rejection (ACR) at 3 months (ACR-3) post-transplant. We performed RNA sequencing on whole blood collected from 88 individuals at the time of 3-month surveillance biopsy to identify transcripts associated with ACR-3, developed a novel sequencing-based targeted expression assay, and validated this gene signature in an independent cohort.

Results
Study participants with ACR-3 had significantly higher risk than those without ACR-3 of subsequent clinical acute rejection at 12 and 24 months, faster decline in graft function, and decreased graft survival in adjusted Cox analysis. We identified a 17-gene signature in peripheral blood that accurately diagnosed ACR-3, and validated it using microarray expression profiles of blood samples from 65 transplant recipients in the GoCAR cohort and three public microarray datasets. In an independent cohort of 110 transplant recipients, tests of the targeted expression assay on the basis of the 17-gene set showed that it identified individuals at higher risk of ongoing acute rejection and future graft loss.

Conclusions
Our targeted expression assay enabled noninvasive diagnosis of subclinical acute rejection and inflammation in the graft and may represent a useful tool to risk-stratify kidney transplant recipients.