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A Peripheral Blood Gene Expression Signature to Diagnose Subclinical Acute Rejection

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A Peripheral Blood Gene Expression Signature to Diagnose Subclinical Acute Rejection CLINICAL RESEARCH www.jasn.org A Peripheral Blood Gene Expression Signature to Diagnose Subclinical Acute Rejection Weijia Zhang,1 Zhengzi Yi,1 Karen L. Keung,2 Huimin Shang,3 Chengguo Wei,1 Paolo Cravedi,1 Zeguo Sun,1 Caixia Xi,1 Christopher Woytovich,1 Samira Farouk,1 Weiqing Huang,1 Khadija Banu,1 Lorenzo Gallon,4 Ciara N. Magee,5 Nader Najafian,5 Milagros Samaniego,6 Arjang Djamali ,7 Stephen I. Alexander,2 Ivy A. Rosales,8 Rex Neal Smith,8 Jenny Xiang,3 Evelyne Lerut,9 Dirk Kuypers,10,11 Maarten Naesens ,10,11 Philip J. O’Connell,2 Robert Colvin,8 Madhav C. Menon,1 and Barbara Murphy1 Due to the number of contributing authors, the affiliations are listed at the end of this article. ABSTRACT 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 indi- viduals 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 subse- quent 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 trans- plant 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. JASN 30: 1481–1494, 2019. doi: https://doi.org/10.1681/ASN.2018111098 CLINICAL RESEARCH Kidney transplantation is the therapy of choice for ESRD. Although short-term allograft outcomes Received November 8, 2018. Accepted May 1, 2019. including clinical acute rejection episodes (i.e., occurring in the presence of graft dysfunction) Published online ahead of print. Publication date available at www.jasn.org. have declined over past decades, proportionate im- provement in long-term allograft survival remains Correspondence: Dr. Barbara Murphy, Division of Nephrology, Department of Medicine, Icahn School of Medicine at Mount 1–3 unrealized. The rates of clinically detected epi- Sinai, One Gustave L Levy Place, Box 1243, New York, NY 10029. sodes of acute rejection within the first year in the Email: [email protected] modern tacrolimus/mycophenolate era are ,10% Copyright © 2019 by the American Society of Nephrology JASN 30: 1481–1494, 2019 ISSN : 1046-6673/3008-1481 1481 CLINICAL RESEARCH www.jasn.org among adult kidney recipients in the United States.2 The effect Significance Statement of these episodes on long-term graft survival is variable, de- pending on the severity of episode, time from transplantation, Biomarkers for noninvasive diagnosis of subclinical acute rejection and effective treatment of these episodes with return of creat- are needed to enable risk-stratification and tailoring of immuno- inine to prerejection baseline.4 suppression for kidney transplant recipients. Using RNA sequencing fl analyses of whole blood collected from a cohort of transplant re- Graft in ammation, however, may also occur initially in the cipients at the time of surveillance biopsy, the authors identified a absence of graft function decline. The wide prevalence of sub- transcriptional signature on the basis of a set of 17 genes that ac- clinical rejection, i.e., lymphocytic tubulitis and interstitial in- curately detects ongoing subclinical rejection. After extensive val- flammation in early surveillance biopsy specimens, and its idation, they developed a sequencing-based targeted expression effect on long-term allograft histology, function, and survival 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 5–10 has been appreciated only recently. The effect of milder in an independent cohort of 110 patients. This assay represents a subclinical inflammation, i.e., suspicious or borderline le- potentially useful tool to monitor kidney transplant recipients and sions, on allograft outcomes is even less characterized.11 optimize immunosuppressive therapy, although larger studies are In current clinical practice, the diagnosis of either clinical or needed to validate the assay’s clinical utility. subclinical allograft rejection requires a biopsy, a procedure burdened by clinical risks and costs. To obviate these issues, readings .1000 were taken as positive. The surveillance biopsy fi prior studies have tested noninvasive pro ling of urinary pro- specimens in the patients in the Belgian cohort were taken at the 12,13 14–16 teins and blood transcriptomic signatures, but results same time points (3, 6, 12, and 24 months) as for the GoCAR have been inconsistent so far. An assay that could be used in cohort. Highly sensitized patients requiring desensitization were clinical practice for the purpose of accurately diagnosing sub- excluded from the GoCAR study and not present in the Belgium clinical rejection offers the potential to identify and treat un- cohort. We used United Network for Organ Sharing and The derlying subclinical rejection without the need for a biopsy. Australia and New Zealand Dialysis and Transplant Registry da- Herein, we examined the incidence of subclinical rejection tabases to determine long-term outcomes for the GoCAR and borderline lesions over time in the Genomics of Chronic enrollees. Allograft Rejection study (GoCAR), a prospective, multicenter center study in which kidney transplant recipients underwent Genomic Experiments and Data Analysis serial surveillance biopsies. We determined the effect of sub- The details regarding genomic experiments (RNA sequencing, clinical graft inflammation on allograft function and survival, microarray, and targeted RNA expression [TREx] assay) are and developed a clinically applicable assay that detects sub- provided in the Supplemental Material and the data analysis clinical acute rejection by measuring the transcriptome in pe- workflow is depicted in Supplemental Figure 1. Briefly, mRNA ripheral blood. sequencing (Illumina HiSeq4000 sequencer) was performed on 88 samples obtained at 3 months post-transplant in the GoCAR cohort as discovery set for identification of gene sig- METHODS natures associated with ACR-3. After read quality control, mapping, and normalization steps on the raw sequencing Patients reads, the expression data were compared between ACR-3 The study included participants of the GoCAR study and kidney and non-ACR at 3 months (NACR-3), with both induction transplant recipients prospectively followed-up at the University therapy and deceased donor as confounders, to identify dif- Hospitals Leuven, Leuven, Belgium. The GoCAR study is a pro- ferentially expressed genes (DEGs) with a P value ,0.05 using spective,multicenterstudy(UnitedStatesandAustralia)aimedat unpaired LIMMA test,20 a linear model to assess differential investigating the genetics and genomics associated with the de- gene expression in the context of multiple variables. The DEGs velopment of allograft rejection or injury in kidney transplant were then subjected to enrichment analysis for canonical recipients. Patients underwent surveillance biopsies pretrans- pathway, gene ontology, and immune cell types to identify plant (before implantation), and at 3, 6, 12, and 24 months after classes or groups of genes that were associated with ACR. transplant. Patients were followed up for at least 5 years or until Next, a more focused gene set associated with ACR-3 was death. The details of patient enrollment criteria and study design identified from the DEGs using a randomization approach have been previously described.17,18 All biopsy specimens were described previously.18. An optimal gene set with the highest reported for Banff component scores by a central core laboratory AUC (area under the receiver operating characteristic curve) at Massachusetts General Hospital. The diagnosis of acute cel- score for diagnosis of ACR-3 was then determined by fitting a lular rejection (ACR) at 3 months (ACR-3) was made by apply- penalized logistic regression model on the expression data of ing Banff 2013 Classification19 on all clinically indicated and the focus gene set following a 5000-iterations methodology18 surveillance biopsy samples and included borderline subclinical (Supplemental Material). The AUC for the final gene set was rejection. Donor-specific anti-HLA antibodies
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