Prediction of Liver Transplant Rejection With a Biologically Relevant Gene Expression Signature

Josh Levitsky, Manoj Kandpal, Kexin Guo, Lihui Zhao, Sunil Kurian, Thomas Whisenant, Michael Abecassis, Josh Levitsky, Manoj Kandpal, Kexin Guo, Lihui Zhao, Sunil Kurian, Thomas Whisenant, Michael Abecassis

Abstract

Background: Noninvasive biomarkers distinguishing early immune activation before acute rejection (AR) could more objectively inform immunosuppression management in liver transplant recipients (LTRs). We previously reported a genomic profile distinguishing LTR with AR versus stable graft function. This current study includes key phenotypes with other causes of graft dysfunction and uses a novel random forest approach to augment the specificity of predicting and diagnosing AR.

Methods: Gene expression results in LTRs with AR versus non-AR (combination of other causes of graft dysfunction and normal function) were analyzed from single and multicenter cohorts. A 70:30 approach (61 ARs; 162 non-ARs) was used for training and testing sets. Microarray data were normalized using a LT-specific vector.

Results: Random forest modeling on the training set generated a 59-probe classifier distinguishing AR versus non-AR (area under the curve 0.83; accuracy 0.78, sensitivity 0.70, specificity 0.81, positive predictive value 0.54, negative predictive value [NPV] 0.89; F-score 0.61). Using a locked threshold, the classifier performed well on the testing set (accuracy 0.72, sensitivity 0.67, specificity 0.73, positive predictive value 0.48, NPV 0.86; F-score 0.56). Probability scores increased in samples preceding AR versus non-AR, when liver function tests were normal, and decreased following AR treatment (P < 0.001). Ingenuity pathway analysis of the genes revealed a high percentage related to immune responses and liver injury.

Conclusions: We have developed a blood-based biologically relevant biomarker that can be detected before AR-associated graft injury distinct from LTR never developing AR. Given its high NPV ("rule out AR"), the biomarker has the potential to inform precision-guided immunosuppression minimization in LTRs.

Conflict of interest statement

J.L. and S.K. provided consultancy to Transplant Genomics Incorporated (Eurofins/Viracor). M.A. is a cofounder of Transplant Genomics Incorporated (Eurofins/Viracor). J.L. received research funding from Novartis. The other authors declare no conflicts of interest.

Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.

Figures

FIGURE 1.
FIGURE 1.
The ROCs—AR vs non-AR. The AUC is displayed as well as the performance characteristics (25% AR prevalence adjustment) at the 0.34 threshold. AR, acute rejection; AUC, area under the curve; ROC, receiver operating curve.
FIGURE 2.
FIGURE 2.
Serial changes in AR vs non-AR gene expression scores using line slopes. A, Pre-AR vs pre–non-AR (P = 0.0002). B, Pre-AR vs post-AR (P = 0.0001). AR, acute rejection.
FIGURE 3.
FIGURE 3.
Three-dimensional principal component analysis score plots for sample clustering, using 59 probes from the random forest classifier between AR and non-AR. The left plot is for training (n = 156) and the right plot is for testing (67) sample sets. AR, acute rejection.

References

    1. Charlton M, Seaberg E. Impact of immunosuppression and acute rejection on recurrence of hepatitis C: results of the National Institute of Diabetes and Digestive and Kidney Diseases Liver Transplantation Database. Liver Transpl Surg. 1999;5:S107–S114.
    1. Wiesner RH, Demetris AJ, Belle SH, et al. . Acute hepatic allograft rejection: incidence, risk factors, and impact on outcome. Hepatology. 1998;28:638–645.
    1. US Multicenter FK506 Liver Study Group. A comparison of tacrolimus (FK 506) and cyclosporine for immunosuppression in liver transplantation. N Engl J Med. 1994;331:1110–1115.
    1. Starzl TE, Klintmalm GB, Porter KA, et al. . Liver transplantation with use of cyclosporin a and prednisone. N Engl J Med. 1981;305:266–269.
    1. Levitsky J, Goldberg D, Smith AR, et al. . Acute rejection increases risk of graft failure and death in recent liver transplant recipients. Clin Gastroenterol Hepatol. 2017;15:584–593.e2.
    1. Demetris AJ, Bellamy C, Hübscher SG, et al. . 2016 comprehensive update of the Banff Working Group on Liver Allograft Pathology: introduction of antibody-mediated rejection. Am J Transplant. 2016;16:2816–2835.
    1. Charlton M, Levitsky J, Aqel B, et al. . International liver transplantation society consensus statement on immunosuppression in liver transplant recipients. Transplantation. 2018;102:727–743.
    1. VanWagner LB, Serper M, Kang R, et al. . Factors associated with major adverse cardiovascular events after liver transplantation among a national sample. Am J Transplant. 2016;16:2684–2694.
    1. Rubín A, Sánchez-Montes C, Aguilera V, et al. . Long-term outcome of ‘long-term liver transplant survivors’. Transpl Int. 2013;26:740–750.
    1. Watt KDS, Pedersen RA, Kremers WK, et al. . Evolution of causes and risk factors for mortality post-liver transplant: results of the NIDDK long-term follow-up study. Am J Transplant. 2010;10:1420–1427.
    1. Levitsky J, O’Leary JG, Asrani S, et al. . Protecting the kidney in liver transplant recipients: practice-based recommendations from the American Society of Transplantation Liver and Intestine Community of Practice. Am J Transplant. 2016;16:2532–2544.
    1. Kourkoumpetis T, Levitsky J. Immunosuppressive drug levels in liver transplant recipients: impact in decision making. Semin Liver Dis. 2019;39:414–421.
    1. Levitsky J. Next level of immunosuppression: drug/immune monitoring. Liver Transpl. 2011;17(suppl 3):S60–S65.
    1. Kowalski RJ, Post DR, Mannon RB, et al. . Assessing relative risks of infection and rejection: a meta-analysis using an immune function assay. Transplantation. 2006;82:663–668.
    1. Xue F, Zhang J, Han L, et al. . Immune cell functional assay in monitoring of adult liver transplantation recipients with infection. Transplantation. 2010;89:620–626.
    1. Fan H, Li L-X, Han D-D, et al. . Increase of peripheral Th17 lymphocytes during acute cellular rejection in liver transplant recipients. Hepatobiliary Pancreat Dis Int. 2012;11:606–611.
    1. Farid WRR, Pan Q, van der Meer AJP, et al. . Hepatocyte-derived microRNAs as serum biomarkers of hepatic injury and rejection after liver transplantation. Liver Transpl. 2012;18:290–297.
    1. Gómez-Mateo J, Marin L, López-Alvarez MR, et al. . TGF-beta1 gene polymorphism in liver graft recipients. Transpl Immunol. 2006;17:55–57.
    1. Joshi D, Salehi S, Brereton H, et al. . Distinct microRNA profiles are associated with the severity of hepatitis C virus recurrence and acute cellular rejection after liver transplantation. Liver Transpl. 2013;19:383–394.
    1. Kamei H, Masuda S, Nakamura T, et al. . Association of transporter associated with antigen processing (TAP) gene polymorphisms in donors with acute cellular rejection in living donor liver transplantation. J Gastrointestin Liver Dis. 2013;22:167–171.
    1. Karimi MH, Daneshmandi S, Pourfathollah AA, et al. . Association of IL-6 promoter and IFN-γ gene polymorphisms with acute rejection of liver transplantation. Mol Biol Rep. 2011;38:4437–4443.
    1. Massoud O, Heimbach J, Viker K, et al. . Noninvasive diagnosis of acute cellular rejection in liver transplant recipients: a proteomic signature validated by enzyme-linked immunosorbent assay. Liver Transpl. 2011;17:723–732.
    1. Moya-Quiles MR, Alvarez R, Miras M, et al. ; Spanish Group of Transplant Immunology and Immunotolerance (G03/104). Impact of recipient HLA-C in liver transplant: a protective effect of HLA-Cw*07 on acute rejection. Hum Immunol. 2007;68:51–58.
    1. Sindhi R, Higgs BW, Weeks DE, et al. . Genetic variants in major histocompatibility complex-linked genes associate with pediatric liver transplant rejection. Gastroenterology. 2008;135:830–839.e10.
    1. Asaoka T, Kato T, Marubashi S, et al. . Differential transcriptome patterns for acute cellular rejection in recipients with recurrent hepatitis C after liver transplantation. Liver Transpl. 2009;15:1738–1749.
    1. Gehrau R, Maluf D, Archer K, et al. . Molecular pathways differentiate hepatitis C virus (HCV) recurrence from acute cellular rejection in HCV liver recipients. Mol Med. 2011;17:824–833.
    1. Sreekumar R, Rasmussen DL, Wiesner RH, et al. . Differential allograft gene expression in acute cellular rejection and recurrence of hepatitis C after liver transplantation. Liver Transpl. 2002;8:814–821.
    1. Shaked A, Chang B-L, Barnes MR, et al. . An ectopically expressed serum miRNA signature is prognostic, diagnostic, and biologically related to liver allograft rejection. Hepatology. 2017;65:269–280.
    1. Bonaccorsi-Riani E, Pennycuick A, Londoño M-C, et al. . Molecular characterization of acute cellular rejection occurring during intentional immunosuppression withdrawal in liver transplantation. Am J Transplant. 2016;16:484–496.
    1. Levitsky J, Asrani SK, Schiano T, et al. ; Clinical Trials in Organ Transplantation - 14 Consortium. Discovery and validation of a novel blood-based molecular biomarker of rejection following liver transplantation. Am J Transplant. 2020;20:2173–2183.
    1. Banff schema for grading liver allograft rejection: an international consensus document. Hepatology. 1997;25:658–663.
    1. Londoño M-C, Souza LN, Lozano J-J, et al. . Molecular profiling of subclinical inflammatory lesions in long-term surviving adult liver transplant recipients. J Hepatol. 2018;69:626–634.
    1. Feng S, Bucuvalas JC, Demetris AJ, et al. . Evidence of chronic allograft injury in liver biopsies from long-term pediatric recipients of liver transplants. Gastroenterology. 2018;155:1838–1851.e7.
    1. Michell CM, Nass SJ, Ommen GS; Institute of Medicine. Evolution of Translational Omics: Lessons Learned and the Path Forward. The National Academies Press; 2012.
    1. Friedewald JJ, Kurian SM, Heilman RL, et al. ; Clinical Trials in Organ Transplantation 08 (CTOT-08). Development and clinical validity of a novel blood-based molecular biomarker for subclinical acute rejection following kidney transplant. Am J Transplant. 2019;19:98–109.
    1. Kurian SM, Williams AN, Gelbart T, et al. . Molecular classifiers for acute kidney transplant rejection in peripheral blood by whole genome gene expression profiling. Am J Transplant. 2014;14:1164–1172.
    1. McCall MN, Bolstad BM, Irizarry RA. Frozen robust multiarray analysis (fRMA). Biostatistics. 2010;11:242–253.
    1. Kurian SM, Whisenant T, Mas V, et al. . Biomarker guidelines for high-dimensional genomic studies in transplantation: adding method to the madness. Transplantation. 2017;101:457–463.
    1. Tibshirani R, Hastie T, Narasimhan B, et al. . Diagnosis of multiple cancer types by shrunken centroids of gene expression. Proc Natl Acad Sci U S A. 2002;99:6567–6572.
    1. Thévenot EA, Roux A, Xu Y, et al. . Analysis of the human adult urinary metabolome variations with age, body mass index, and gender by implementing a comprehensive workflow for univariate and OPLS statistical analyses. J Proteome Res. 2015;14:3322–3335.
    1. Meyer D, Dimitriadou E, Hornik K, et al. . e1071: Misc Functions of the Department of Statistics, Probability Theory Group (Formerly: E1071), TU Wien. Available at . Accessed May 23, 2021.
    1. Liaw A, Wiener M. Classification and regression by randomForest. R News. 2002;2:18–22.
    1. Friedman J, Hastie T, Tibshirani R. Regularization paths for generalized linear models via coordinate descent. J Stat Softw. 2010;33:1–22.
    1. Van Loon E, Gazut S, Yazdani S, et al. . Development and validation of a peripheral blood mRNA assay for the assessment of antibody-mediated kidney allograft rejection: a multicentre, prospective study. EBioMedicine. 2019;46:463–472.
    1. Ramji A, Yoshida EM, Bain VG, et al. . Late acute rejection after liver transplantation: the Western Canada experience. Liver Transpl. 2002;8:945–951.
    1. Uemura T, Ikegami T, Sanchez EQ, et al. . Late acute rejection after liver transplantation impacts patient survival. Clin Transplant. 2008;22:316–323.
    1. Thurairajah PH, Carbone M, Bridgestock H, et al. . Late acute liver allograft rejection; a study of its natural history and graft survival in the current era. Transplantation. 2013;95:955–959.
    1. Toby TK, Abecassis M, Kim K, et al. . Proteoforms in peripheral blood mononuclear cells as novel rejection biomarkers in liver transplant recipients. Am J Transplant. 2017;17:2458–2467.
    1. Shaked A, DesMarais MR, Kopetskie H, et al. . Outcomes of immunosuppression minimization and withdrawal early after liver transplantation. Am J Transplant. 2019;19:1397–1409.
    1. Jucaud V, Shaked A, DesMarais M, et al. . Prevalence and impact of de novo donor-specific antibodies during a multicenter immunosuppression withdrawal trial in adult liver transplant recipients. Hepatology. 2019;69:1273–1286.
    1. Pontier DB, Gribnau J. Xist regulation and function explored. Hum Genet. 2011;130:223–236.
    1. Shen C, Li J. LncRNA XIST silencing protects against sepsis-induced acute liver injury via inhibition of BRD4 expression. Inflammation. 2021;44:194–205.
    1. Cheng Q, Wang L. LncRNA XIST serves as a ceRNA to regulate the expression of ASF1A, BRWD1M, and PFKFB2 in kidney transplant acute kidney injury via sponging hsa-miR-212-3p and hsa-miR-122-5p. Cell Cycle. 2020;19:290–299.
    1. Grozdanov V, Danzer KM. Intracellular alpha-synuclein and immune cell function. Front Cell Dev Biol. 2020;8:562692.
    1. Shabtai M, Waltzer WC, Ayalon A, et al. . Down regulation of CD45 expression on CD4 T cells during acute renal allograft rejection: evidence of a decline in T suppressor/inducer activity. Int Urol Nephrol. 2002;34:555–558.
    1. Hendriks J, Xiao Y, Borst J. CD27 promotes survival of activated T cells and complements CD28 in generation and establishment of the effector T cell pool. J Exp Med. 2003;198:1369–1380.
    1. Kearns GL, Artman M. Functional biomarkers: an approach to bridge pharmacokinetics and pharmacodynamics in pediatric clinical trials. Curr Pharm Des. 2015;21:5636–5642.
    1. Modena BD, Kurian SM, Gaber LW, et al. . Gene expression in biopsies of acute rejection and interstitial fibrosis/tubular atrophy reveals highly shared mechanisms that correlate with worse long-term outcomes. Am J Transplant. 2016;16:1982–1998.
    1. Mengel M, Sis B, Kim D, et al. . The molecular phenotype of heart transplant biopsies: relationship to histopathological and clinical variables. Am J Transplant. 2010;10:2105–2115.
    1. Pham MX, Teuteberg JJ, Kfoury AG, et al. ; IMAGE Study Group. Gene-expression profiling for rejection surveillance after cardiac transplantation. N Engl J Med. 2010;362:1890–1900.
    1. Soma O, Hatakeyama S, Yoneyama T, et al. . Serum N-glycan profiling can predict biopsy-proven graft rejection after living kidney transplantation. Clin Exp Nephrol. 2020;24:174–184.
    1. Bohne F, Martínez-Llordella M, Lozano J-J, et al. . Intra-graft expression of genes involved in iron homeostasis predicts the development of operational tolerance in human liver transplantation. J Clin Invest. 2012;122:368–382.
    1. Bien-Willner GA. Biomarkers and cancer: correlation is not causation. Available at . Accessed January 6, 2015.
    1. Mayeux R. Biomarkers: potential uses and limitations. NeuroRx. 2004;1:182–188.
    1. Steyerberg EW, Harrell FE, Jr. Prediction models need appropriate internal, internal-external, and external validation. J Clin Epidemiol. 2016;69:245–247.
    1. Steyerberg EW, Bleeker SE, Moll HA, et al. . Internal and external validation of predictive models: a simulation study of bias and precision in small samples. J Clin Epidemiol. 2003;56:441–447.
    1. Harrell FE, Jr, Lee KL, Mark DB. Tutorial in biostatistics. Multivariable prognostic models: issues in developing models, evaluating assumptions and adequacy, and measuring and reducing errors. Stat Med. 1996;15:361–387.
    1. O’Leary JG, Demetris AJ, Friedman LS, et al. . The role of donor-specific HLA alloantibodies in liver transplantation. Am J Transplant. 2014;14:779–787.
    1. O’Leary JG, Kaneku H, Jennings LW, et al. . Preformed class II donor-specific antibodies are associated with an increased risk of early rejection after liver transplantation. Liver Transpl. 2013;19:973–980.
    1. Levitsky J, Kaneku H, Jie C, et al. . Donor-specific HLA antibodies in living versus deceased donor liver transplant recipients. Am J Transplant. 2016;16:2437–2444.

Source: PubMed

Спонсоры и соавторы

Заболевания

Медикаментозные вмешательства

Подписаться