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Current Status of Biomarkers and Molecular Diagnostic Tools for Rejection in Liver Transplantation: Light at the End of theTunnel?

      Strategies to minimize immune-suppressive medications after liver transplantation are limited by allograft rejection. Biopsy of liver is the current standard of care in diagnosing rejection. However, it adds to physical and economic burden to the patient and has diagnostic limitations. In this review, we aim to highlight the different biomarkers to predict and diagnose acute rejection. We also aim to explore recent advances in molecular diagnostics to improve the diagnostic yield of liver biopsies.

      Graphical abstract

      Keywords

      Abbreviations:

      LT (Liver transplantation), UNOS (United network for organ sharing and procurement), ILTS (International liver transplantation society), CNI (Calcineurin inhibitors), MToR (Mechanistic target of Rapamycin), MMF (Mycophenolate Mofetil), ATG (Anti-thymoglobulin), GLUT-4 (glucose transport-4), ATCMR (acute T-cell mediated rejection), AMR (antibody mediated rejection), APC (antigen presenting cells), MHC (major histo–compatibility complex), FDA (Food and drug administration), AR (Acute rejection), miRNA (micro-RNA), dd cfDNA (donor-derived cell-free DNA), HNMR (high nuclear magnetic resonance), mRNA (messenger RNA), AUC (area under curve), AUROC (area under receiver operating characteristic curve), PPV (Positive predictive value), NPV (Negative predictive value), DSA (Donor specific antibodies), HLA (human leukocyte antigens), MFI (mean fluorescence intensity), MMDX (Molecular microscopic diagnostic system), RATs (rejection associated transcripts), TBB (trans-bronchial biopsies), 3BMBs (third bifurcation mucosal endo-bronchial biopsies), FFPE (formalin fixed paraffin embedded preparation), B-HOT (Banff Human Organ Transplant), MDWG (molecular diagnostic work group)
      LT represents the only curative option for decompensated liver disease.
      • Shimamura T.
      • Goto R.
      • Watanabe M.
      • Kawamura N.
      • Takada Y.
      Liver transplantation for hepatocellular carcinoma: how should we improve the thresholds?.
      According to United network for organ sharing and procurement (UNOS), 8869 LTs were performed in the US in 2019. One of the challenges that affect graft survival and overall mortality in transplanted patients is graft rejection.
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      • Goldberg D.
      • Smith A.R.
      • et al.
      Acute rejection increases risk of graft failure and death in recent liver transplant recipients.
      Liver biopsy is considered the SOC to diagnose acute rejection and serves as the cornerstone for therapeutic interventions. However, it is invasive, expensive, and may result in complications. Also, its interpretation may be difficult in the presence of infection or recurrence of primary disease. There is a need of non-invasive biomarkers to predict the onset and severity of acute rejection and improvement in histological markers to make an accurate diagnosis.
      • Krenzien F.
      • Keshi E.
      • Splith K.
      • et al.
      Diagnostic biomarkers to diagnose acute allograft rejection after liver transplantation: systematic review and meta-analysis of diagnostic accuracy studies.
      In this review, we aim to explore newer biomarkers and advances in molecular diagnostics and their promising use in patients with LT.

      Current state of immune-suppression in liver transplantation

      Transplantation of any solid organ induces a T-cell-mediated immune response, which might lead to graft rejection. Although liver has a special micro-environment that promotes tolerance, immune-suppressive (IS) medications are still necessary to avoid graft rejection and failure.
      • Sánchez–Fueyo A.
      • Strom T.B.
      Immunologic basis of graft rejection and tolerance following transplantation of liver or other solid organs.
      They are used to induce and maintain graft tolerance, and also to treat rejection.
      • Moini M.
      • Schilsky M.L.
      • Tichy E.M.
      Review on immunosuppression in liver transplantation.
      According to the latest consensus statement by the international liver transplantation society (ILTS), many regimens are used as IS agents in LT, and include one or more of the following pharmacological classes:
      • Corticosteroids
      • Calcineurin inhibitors (CNIs): Tacrolimus and cyclosporine
      • Mechanistic target of Rapamycin (MTOR) inhibitors: Sirolimus and everolimus
      • Anti-metabolites: Mycophenolate mofetil and azathioprine
      • Depleting monoclonal antibodies: Anti-thymoglobulin and alemtuzumab
      • Non-depleting monoclonal antibodies: Basiliximab
        • Charlton M.
        • Levitsky J.
        • Aqel B.
        • et al.
        International liver transplantation society consensus statement on immunosuppression in liver transplant recipients.
      Each class and specific agent has unique efficacy and side–effect profile, and a tailored immune-suppression is essential for the best outcome after transplantation.
      • Moini M.
      • Schilsky M.L.
      • Tichy E.M.
      Review on immunosuppression in liver transplantation.
      ,
      • Di Maira T.
      • Little E.C.
      • Berenguer M.
      Immunosuppression in liver transplant.

      Challenges with immune-suppression

      Long-term follow-up of liver transplant patients shows significant morbidity and all-cause mortality related to immune-suppression. Infection is the most common cause of death in the first year post-LT. Malignancy, cardiovascular complications, and renal impairment constitute the main causes of death in later years.
      • Watt K.D.S.
      • Pedersen R.A.
      • Kremers W.K.
      • Heimbach J.K.
      • Charlton M.R.
      Evolution of causes and risk factors for mortality post-liver transplant: results of the NIDDK long-term follow-up study.
      CNIs are associated with diabetes mellitus due to their effects on glucose transport-4 thus increasing insulin resistance and also affecting β-cell functions, while MTOR inhibitors are associated with hypertriglyceridemia and metabolic syndrome evolution.
      • Azhie A.
      • Sheth P.
      • Hammad A.
      • Woo M.
      • Bhat M.
      Metabolic complications in liver transplantation recipients: how we can optimize long-term survival.
      Chronic kidney disease resulting from hyaline arteriolosclerosis, glomerulo-sclerosis, and tubule-interstitial nephritis is a recognized risk in up to 70% of LT patients with chronic CNIs.
      • Beckebaum S.
      • Cicinnati V.R.
      • Radtke A.
      • Kabar I.
      Calcineurin inhibitors in liver transplantation – still champions or threatened by serious competitors?.
      ,
      • Ziolkowski J.
      • Paczek L.
      • Senatorski G.
      • et al.
      Renal function after liver transplantation: calcineurin inhibitor nephrotoxicity.
      Multiple reports showed that immune-suppression is linked to development of skin cancers (squamous cell carcinoma and basal cell carcinoma), lymphoproliferative disorders, colorectal carcinoma, and lung cancers in LT patients.
      • Chak E.
      • Saab S.
      Risk factors and incidence of de novo malignancy in liver transplant recipients: a systematic review: risk factors and incidence of de novo malignancy in liver transplant recipients.
      The complications of immune-suppression are dependent on dose, class, and duration of the drugs. Therefore, reduction in immune-suppression is helpful in most patients starting early following transplantation and a full withdrawal of immune-suppression may be possible in select patients.
      • Charlton M.
      • Levitsky J.
      • Aqel B.
      • et al.
      International liver transplantation society consensus statement on immunosuppression in liver transplant recipients.
      However, strategies to minimize or withdraw immune-suppression have been limited by acute graft rejection. Shaked et al. reported that 42 out of 52 patients did not tolerate reduction of immune-suppression dose to less than 50% of the baseline due to development of acute rejection.
      • Shaked A.
      • DesMarais M.R.
      • Kopetskie H.
      • et al.
      Outcomes of immunosuppression minimization and withdrawal early after liver transplantation.
      Figure 1 shows that balancing immune-suppression to avoid complications is necessary; however, present tools are inaccurate in evaluating the state of immune-suppression. Future direction focuses on biomarkers and molecular diagnostics to accurately identify the underlying pathways of liver rejection.
      Figure 1
      Figure 1Current and future strategies to adjust immune-suppression.

      Rejection and graft survival

      Rejection in LT is a major cause of morbidity and may lead to graft failure and mortality. Graft rejection may present as acute T-cell-mediated rejection (ATCMR), antibody-mediated rejection (AMR), and chronic rejection. Each subtype has different pathophysiological characteristics and clinical presentation and requires different management modalities.
      • Levitsky J.
      • Goldberg D.
      • Smith A.R.
      • et al.
      Acute rejection increases risk of graft failure and death in recent liver transplant recipients.
      ATCMR occurs in 15–25% of LT patients in the first year following LT. It presents as elevated aminotransferases, bilirubin, and cholestatic enzymes, it may be asymptomatic in 7–11% of patients, especially after 1 year post-transplantation.
      • Nacif L.S.
      • Pinheiro R.S.
      • Pécora RAdA.
      • et al.
      Late acute rejection in liver transplant: a systematic review.
      • Thurairajah P.H.
      • Carbone M.
      • Bridgestock H.
      • et al.
      Late acute liver allograft rejection; a study of its natural history and graft survival in the current era.
      • Akamatsu N.
      • Sugawara Y.
      • Tamura S.
      • et al.
      Late-onset acute rejection after living donor liver transplantation.
      • Hutchinson J.A.
      • Schlitt H.J.
      Subclinical T cell-mediated liver transplant rejection: the jury is still out.
      ATCMR has two overlapping phases: allograft recognition and effector responses. Allograft recognition has 3 pathways (Figure 2): (1) direct pathway in which the donor antigen presenting cells (APCs) migrate to the lymphoid tissues where the recipient naïve CD4+ T-lymphocyte recognizes both the presented antigen and the major histo–compatibility complex (MHC) class II on donor APCs as allogeneic. Similarly, donor class I MHC molecules also are recognized by CD8+ T-cell.
      • Demetris A.J.
      • Bellamy C.O.
      • Gandhi C.R.
      • Prost S.
      • Nakanuma Y.
      • Stolz D.B.
      Functional immune anatomy of the liver-as an allograft.
      (2) Indirect pathway in which the recipient APCs present phagocytosed allogeneic material shed by donor cells to the recipient CD4+ T-helper cells. (3) Semi-indirect pathway in which a transfer of donor MHC molecules from donor APCs to recipient dendritic cell occurs by exocytosis, and then, antigen presentation takes place through the recipient APCs.
      • Ronca V.
      • Wootton G.
      • Milani C.
      • Cain O.
      The immunological basis of liver allograft rejection.
      These pathways lead to naïve CD4+ T-cell to differentiate to Th1, Th2, Th17, and Reg-T-cells and migration of both CD4+ T-cells and CD8+ cells to the liver graft.
      • Smyth L.A.
      • Lechler R.I.
      • Lombardi G.
      Continuous acquisition of MHC:peptide complexes by recipient cells contributes to the generation of anti-graft CD8(+) T cell immunity.
      The second phase involves cellar damaged by the recruited T-cells. CD8+ T-cell is the main mediator of the direct cytotoxic damage via perforin/granzyme pathway and FAS/FASL pathway leading to direct hepatocyte damage, aided by the various cytokines produced by CD4+ T-cell subtypes which include IL-1, IL-2, IL-4, IL-17, TNF-α, and INF-γ.
      • Ronca V.
      • Wootton G.
      • Milani C.
      • Cain O.
      The immunological basis of liver allograft rejection.
      Figure 2
      Figure 2Allograft recognition pathways and immune effector pathways. 3 pathways has been identified; in direct pathway donor APCs, MHC class II molecules are recognized by recipient CD4+ T-cells as foreign and recipient CD8+ T-cells recognize MHC class I molecules on donor APCs. In indirect pathway, recipient APCs present graft antigen to recipient CD4+ T-cells, semi-indirect pathway combines both pathways as recipient APCs present exocytosis transferred donor MHC molecules to recipient CD4+ T-cells. Effector phase depends on hepatocyte cytotoxicity by activated CD8+ T-cell augmented by cytokines release from CD4+ T-cells.
      AMR is poorly understood in LT, with incidence less than 1% in ABO matched grafts.
      • Demetris A.J.
      • Zeevi A.
      • O'Leary J.G.
      ABO-compatible liver allograft antibody-mediated rejection: an update.
      Banff group defines AMR with the presence of four criteria: (1) endothelial hypertrophy, portal capillary dilatation, micro-vasculitis, and involvement of the central vein; (2) C4d deposition in portal microvascular endothelium; (3) high titer of donor specific antibodies; (4) no other cause to explain the presentation.
      • Demetris A.J.
      • Bellamy C.
      • Hübscher S.G.
      • et al.
      2016 comprehensive update of the Banff working group on liver allograft pathology: introduction of antibody-mediated rejection.
      Chronic graft rejection is a late complication that usually develops months to years after transplantation. It is uncommon complication that occurs in 3.3% of LT recipients,
      • Angelico R.
      • Sensi B.
      • Manzia T.M.
      • et al.
      Chronic rejection after liver transplantation: opening the Pandora's box.
      ,
      • Blakolmer K.
      • Jain A.
      • Ruppert K.
      • et al.
      Chronic liver allograft rejection in a population treated primarily with tacrolimus as baseline immunosuppression: long-term follow-up and evaluation of features for histopathological staging.
      mostly related to non-optimized immune-suppression, characterized by ductopenia and parenchymal fibrosis and may lead to graft failure requiring re-transplantation.
      • Choudhary N.S.
      • Saigal S.
      • Bansal R.K.
      • Saraf N.
      • Gautam D.
      • Soin A.S.
      Acute and chronic rejection after liver transplantation: what A clinician needs to know.
      Graft rejection leads to decreased overall graft survival, increase in hospital admission and mortality, irrespective of the time and type of rejection from transplantation.
      • Levitsky J.
      • Goldberg D.
      • Smith A.R.
      • et al.
      Acute rejection increases risk of graft failure and death in recent liver transplant recipients.
      ,
      • Dogan N.
      • Hüsing-Kabar A.
      • Schmidt H.H.
      • Cicinnati V.R.
      • Beckebaum S.
      • Kabar I.
      Acute allograft rejection in liver transplant recipients: incidence, risk factors, treatment success, and impact on graft failure.
      Currently, there are no direct measures to evaluate the state of immune-suppression in the graft. We monitor IS drug levels and biochemical markers of liver inflammation with elevated liver enzymes and rely on liver biopsy to diagnose rejection as the SOC. However, liver enzymes cannot differentiate between rejection, infection, or biliary obstruction. Biopsies are expensive, time consuming, and carry a risk of bleeding. It led to the search for a more affordable, easier to implement, and safer non-invasive biomarkers. The goal is to predict and diagnose rejection to guide clinical interventions early in the course for a better outcome.
      • Neuberger J.
      • Cain O.
      The need for alternatives to liver biopsies: non-invasive analytics and diagnostics.
      ,
      • Kamali K.
      • Schmelzle M.
      • Kamali C.
      • et al.
      Sensing acute cellular rejection in liver transplant patients using liver-derived extracellular particles: a prospective, observational study. Original research.

      History of biomarkers for rejection

      A biomarker is a measurable quantity that can evaluate a physiological process, assess, and predict a pathological one. Ideally, it needs to be non-invasive, inexpensive, and easy to perform with a high specificity and sensitivity, and reproducible with validated studies.
      • Califf R.M.
      Biomarker definitions and their applications.
      Search of biomarkers to detect liver rejection started 40 years ago with Neopterin, a catabolic product of purine metabolism and a marker of macrophage activity which was the first one to be studied.
      • Margreiter R.
      • Fuchs D.
      • Hausen A.
      • et al.
      Neopterin as a new biochemical marker for diagnosis of allograft rejection: experience based upon evaluation of 100 consecutive cases.
      Further search for biomarkers focused on cytokines and inflammatory markers such as IL-2R, TNF-α, CD-28 expression, IL-15 and β2-micro-globin, biliary markers, and other markers not related to inflammation. However, all of them failed to be implemented into clinical use due to their inability to differentiate ATCMR from other conditions (infection and biliary obstruction) and the absence of validation in large multicenter studies, Figure 3 summarize categories of the these biomarkers.
      • Verhelst X.P.D.
      • Troisi R.I.
      • Colle I.
      • Geerts A.
      • van Vlierberghe H.
      Biomarkers for the diagnosis of acute cellular rejection in liver transplant recipients: a review: biomarkers for ACR in liver transplantation.
      Figure 3
      Figure 3Historic biomarkers of liver rejection.
      ImmuKnow, a marker approved by FDA in 2002, is a molecular assay using intra-cellular concentration of adenosine triphosphate (ATP) in CD4+ T-helper cell to assess immune system activity.
      • Israeli M.
      • Klein T.
      • Sredni B.
      • et al.
      ImmuKnow: a new parameter in immune monitoring of pediatric liver transplantation recipients.
      It was proposed that CD4+ cellular activity correlates with the state of immune-suppression. It can predict rejection with a higher ATP level and infection with a lower level. Despite its initial promising results, pooled results in a meta-analysis failed to show its predictive value in acute rejection with a sensitivity of 0.43, specificity of 0.75, and positive likelihood ratio of 1.30 (95% CI: 0.74–2.28). Another meta-analysis showed that low levels of ATP may identify patients with increased risk of infection; however, no correlation was found with acute rejection.
      • Rodrigo E.
      • López-Hoyos M.
      • Corral M.
      • et al.
      ImmuKnow as a diagnostic tool for predicting infection and acute rejection in adult liver transplant recipients: a systematic review and meta-analysis.
      ,
      • Xiaoting L.
      • Jun X.
      • Wenhua L.
      • et al.
      Can immune cell function assay identify patients at risk of infection or rejection? A meta-analysis.

      Era of new biomarkers

      Advances in molecular biology have led to the evolution of modern fields of biomarkers such as metabolic, transcription factors, and genetic biomarkers.
      • Verhelst X.P.D.
      • Troisi R.I.
      • Colle I.
      • Geerts A.
      • van Vlierberghe H.
      Biomarkers for the diagnosis of acute cellular rejection in liver transplant recipients: a review: biomarkers for ACR in liver transplantation.
      We systematically reviewed the literature to identify prominent studies exploring the new biomarkers, which are summarized in table 1.
      Table 1New Biomarkers of Rejection in Liver Transplantation.
      AuthorBiomarkerSample typepopulationOutcome
      Metabolic biomarkersDuarte
      • Duarte I.F.
      • Stanley E.G.
      • Holmes E.
      • et al.
      Metabolic assessment of human liver transplants from biopsy samples at the donor and recipient stages using high-resolution magic angle spinning 1H NMR spectroscopy.
      Phospholipid and triglyceride contentLiver biopsy6 adultsIdentification of liver graft metabolic profile by HNMR
      Cortes
      • Cortes M.
      • Pareja E.
      • García-Cañaveras J.C.
      • et al.
      Metabolomics discloses donor liver biomarkers associated with early allograft dysfunction.
      Phospholipid, histidine and Bile acid biosynthesisLiver biopsy124 adultsPredicting graft rejection by HNMR
      Transcription FactorsMuthukumar
      • Muthukumar T.
      • Akat K.M.
      • Yang H.
      • et al.
      Serum MicroRNA transcriptomics and acute rejection or recurrent hepatitis C virus in human liver allograft recipients: a pilot study.
      miRNA-22, miR-34a, miRNA-122, miR-148a, miRNA-192, miRNA-193b, miRNA-194, miRNA-210 and miRNA-885-5pSERUM91 adultsDiagnosis of acute rejection, differentiating AR from other inflammatory conditions
      Shaked
      • Shaked A.
      • Chang B.L.
      • Barnes M.R.
      • et al.
      An ectopically expressed serum miRNA signature is prognostic, diagnostic, and biologically related to liver allograft rejection.
      hsa-miRNA-483-3p and hsa-miRNA-885-5pserum69 adultsDifferentiating AR from other inflammatory conditions
      Genetic biomarkersLevitsky
      • Levitsky J.
      • Kandpal M.
      • Guo K.
      • Kleiboeker S.
      • Sinha R.
      • Abecassis M.
      Donor-derived cell-free DNA levels predict graft injury in liver transplant recipients.
      36-gene probeserum186 adultsDiagnosis and prediction of AR
      Goh
      • Goh S.K.
      • Do H.
      • Testro A.
      • et al.
      The measurement of donor-specific cell-free DNA identifies recipients with biopsy-proven acute rejection requiring treatment after liver transplantation.
      dd cfDNASerum20 adultsDiagnosis of AR
      Schütz
      • Schütz E.
      • Fischer A.
      • Beck J.
      • et al.
      Graft-derived cell-free DNA, a noninvasive early rejection and graft damage marker in liver transplantation: a prospective, observational, multicenter cohort study.
      dd cfDNAserum115 adultsEarly detection of AR
      Zhao
      • Zhao D.
      • Zhou T.
      • Luo Y.
      • et al.
      Preliminary clinical experience applying donor-derived cell-free DNA to discern rejection in pediatric liver transplant recipients.
      dd cfDNAserum49 pediatricDiagnosis of AR
      AR, Acute rejection; miR, micro-RNA; dd cfDNA, donor-derived cell-free DNA; HNMR, high nuclear magnetic resonance.

      Metabolic biomarkers

      Metabolic biomarkers depend on detecting multiple metabolic products or intermediary products to assess pathophysiologic processes during liver rejection.
      • Cortes M.
      • García-Cañaveras J.C.
      • Pareja E.
      • Lahoz A.
      Liver transplantation biomarkers in the metabolomics era.
      One of the earliest reports described the use of Hi-nuclear magnetic resonance to evaluate the metabolic profile of grafted liver before and after LT.
      • Duarte I.F.
      • Stanley E.G.
      • Holmes E.
      • et al.
      Metabolic assessment of human liver transplants from biopsy samples at the donor and recipient stages using high-resolution magic angle spinning 1H NMR spectroscopy.
      Another report described the different metabolic patterns associated with graft dysfunction and development of rejection after transplantation.
      • Cortes M.
      • Pareja E.
      • García-Cañaveras J.C.
      • et al.
      Metabolomics discloses donor liver biomarkers associated with early allograft dysfunction.
      Despite their promising prospective, no metabolic profiling technique has yet been transferred to clinical practice.
      The use of fecal microbiota to predict acute rejection is being explored. An ongoing clinical trial is exploring the use of fecal microbiota in patients who underwent living donor LT to predict the development of acute rejection in the first year after transplantation.
      Institute of Liver and Biliary Sciences I
      Role of Fecal Microbiota in Predicting Graft Rejection and Sepsis Among Recipients of Living Donor Liver Transplant in First Year.
      More studies encompassing different cohorts are needed to validate the use of microbiota as a biomarker for acute rejection.

      Transcription factors

      Transcription factors associated with liver rejection is another field of study in evolution. Micro-RNA is a small non-coding RNA sequence regulating transcription processes
      • Mohr A.
      • Mott J.
      Overview of MicroRNA biology.
      and is associated with acute liver rejection by upregulating pro-inflammatory TGF-β pathway and downregulating FOXP3 pathway in regulatory T-cells.
      • Hamdorf M.
      • Kawakita S.
      • Everly M.
      The potential of MicroRNAs as novel biomarkers for transplant rejection.
      A recent study using small-RNA sequencing and miRNA microarray on 91 LT serum samples identified nine micro-RNA sequences that showed statistically significant association with acute rejection and has the ability to distinguish acute rejection from other inflammatory conditions.
      • Muthukumar T.
      • Akat K.M.
      • Yang H.
      • et al.
      Serum MicroRNA transcriptomics and acute rejection or recurrent hepatitis C virus in human liver allograft recipients: a pilot study.
      Another study performed on serum samples of 69 liver recipients and compared to biopsies showed that 2 of the studied micro-RNA sequences were statistically significant in prediction, diagnosis, and differentiating acute rejection from other inflammatory conditions.
      • Shaked A.
      • Chang B.L.
      • Barnes M.R.
      • et al.
      An ectopically expressed serum miRNA signature is prognostic, diagnostic, and biologically related to liver allograft rejection.
      Transcription biomarkers are the only biomarkers being studied with the potential of differentiating acute rejection from other inflammatory causes. Further studies are needed to validate the current results and assess their ability to be transferred to clinical application.

      Genetic biomarkers

      Genetic biomarkers are designed to recognize genomic signaling association with acute rejection and involves both messenger RNA (mRNA) and donor-derived cell-free DNA (dd cfDNA). A recent study has identified a mRNA probe consisting of 36 genes associated with high specificity and negative predictive values (sensitivity 0.57, specificity 0.82, PPV 0.47, and NPV 0.87, AUROC 0.925) in detecting acute rejection. In addition, it showed the ability to predict the occurrence of acute rejection even before development of liver enzyme derangement.
      • Levitsky J.
      • Asrani S.K.
      • Schiano T.
      • et al.
      Discovery and validation of a novel blood-based molecular biomarker of rejection following liver transplantation.
      It needs further validation in randomized trials.
      Donor-derived cell-free DNA (dd cfDNA) is a new promising biomarker under investigation for use to detect cellular rejection in solid organ transplantation. It was hypothesized that shedding of dd cfDNA from graft may correlate with acute rejection. The utility of dd cfDNA has been explored in renal and heart transplant with promising results. In a report of pediatric renal transplantation, dd cfDNA showed sensitivity 86% and specificity 100% in the diagnosis of acute rejection (AUC 0.996)
      • Puliyanda D.P.
      • Swinford R.
      • Pizzo H.
      • Garrison J.
      • De Golovine A.M.
      • Jordan S.C.
      Donor-derived cell-free DNA (dd-cfDNA) for detection of allograft rejection in pediatric kidney transplants.
      and is under investigation in patients with LT.
      • Knight S.R.
      • Thorne A.
      • Lo Faro M.L.
      Donor-specific cell-free DNA as a biomarker in solid organ transplantation. A systematic review.
      In the setting of LT, dd cfDNA studied on 20 patients demonstrated a high sensitivity of 83.3% in detecting acute rejection with AUC 0.98.
      • Goh S.K.
      • Do H.
      • Testro A.
      • et al.
      The measurement of donor-specific cell-free DNA identifies recipients with biopsy-proven acute rejection requiring treatment after liver transplantation.
      Another study showed that dd cfDNA has sensitivity of 90.3% (95% CI 74.2%–98.0%) in the detection of acute rejection and correlates earlier with the development of rejection compared to aminotransferases or cholestatic biomarkers. It also showed that due to a short half-life (<1.5 h), it reflected more accurately with the response to treatment (sensitivity and specificity of 97.1 and 89.3 compared to 95.7 and 82.1 for aminotransferases, respectively).
      • Schütz E.
      • Fischer A.
      • Beck J.
      • et al.
      Graft-derived cell-free DNA, a noninvasive early rejection and graft damage marker in liver transplantation: a prospective, observational, multicenter cohort study.
      To our knowledge, only one report analyzed the diagnostic utility of dd cfDNA in pediatric liver transplant patients. It showed that dd cfDNA yielded specificity of 81.9% and sensitivity of 81.8% in detecting acute rejection; however, its ability to differentiate acute rejection from other causes of liver injury was not explored in that study.
      • Zhao D.
      • Zhou T.
      • Luo Y.
      • et al.
      Preliminary clinical experience applying donor-derived cell-free DNA to discern rejection in pediatric liver transplant recipients.
      Dd cfDNA has a limited ability to accurately differentiate acute rejection from other causes of graft injury such as viral infection (CMV, HCV, HBV), biliary complications, or ischemic injury.
      • Knight S.R.
      • Thorne A.
      • Lo Faro M.L.
      Donor-specific cell-free DNA as a biomarker in solid organ transplantation. A systematic review.
      This limitation is more pronounced in LT than other solid organ transplants. A report of 171 heart transplant recipients showed that dd cfDNA showed a sensitivity of 95% and specificity of 92% and AUROC of 0.92 at a threshold of 0.5%.
      • Agbor-Enoh S.
      • Shah P.
      • Tunc I.
      • et al.
      Cell-free DNA to detect heart allograft acute rejection.
      Another report of 93 renal transplant recipients showed that urinary dd cfDNA has the ability to distinguish acute rejection from viral infection with a sensitivity of 96%, specificity of 91%, and AUROC of 0.745 at a threshold of 0.84%.
      • Chen X.-T.
      • Chen W.-F.
      • Li J.
      • et al.
      Urine donor–derived cell-free DNA helps discriminate BK polyomavirus-associated nephropathy in kidney transplant recipients with BK polyomavirus infection. Original research.
      This disparity may be explained by the ability of hepatocytes to have unlimited proliferation compared to stable cells as in renal parenchyma and cardiac muscle. Consequently, we see higher baseline levels of cfDNA in liver transplant recipients in the absence of hepatocyte damage. In the study by Beck et al., dd cfDNA was <6.8% in stable liver transplant recipients, <2.5% in kidney transplant recipients, and <3.4% in heart transplant recipients.
      • Beck J.
      • Bierau S.
      • Balzer S.
      • et al.
      Digital droplet PCR for rapid quantification of donor DNA in the circulation of transplant recipients as a potential universal biomarker of graft injury.
      Hepatocyte proliferative ability and a higher baseline level of dd cfDNA may explain its limitation to differentiate AR from other causes of liver injury in liver transplant recipients compared to other solid organ recipients.
      • Grskovic M.
      • Hiller D.J.
      • Eubank L.A.
      • et al.
      Validation of a clinical-grade Assay to measure donor-derived cell-free DNA in solid organ transplant recipients.
      None of the available biomarkers has yet reached optimum clinical applicability. Multiple obstacles hinder bench to bedside transition for these biomarkers. As this field is still in its infancy, there are no standardized laboratory methods to evaluate each biomarker, resulting in heterogeneity of study outcomes.
      • Terasaki P.
      • Lachmann N.
      • Cai J.
      Summary of the effect of de novo HLA antibodies on chronic kidney graft failure.
      Variability of expression of the biomarkers between patients and intra-patient variability during the clinical course affect their reliability.
      • Min J.L.
      • Barrett A.
      • Watts T.
      • et al.
      Variability of gene expression profiles in human blood and lymphoblastoid cell lines.
      Furthermore, sample complexity in genetic biomarkers hinders the identification of valid biomarkers. Debey et al., showed that the presence of globin-mRNA complex leads to variability in analyzing gene expression by microarrays.
      • Debey S.
      • Zander T.
      • Brors B.
      • Popov A.
      • Eils R.
      • Schultze J.L.
      A highly standardized, robust, and cost-effective method for genome-wide transcriptome analysis of peripheral blood applicable to large-scale clinical trials.
      ,
      • Roedder S.
      • Vitalone M.
      • Khatri P.
      • Sarwal M.M.
      Biomarkers in solid organ transplantation: establishing personalized transplantation medicine.
      Large prospective studies with well-defined study designs are needed before the acceptance of biomarkers in clinical practice.

      Donor specific antibodies

      Donor-specific antibodies (DSA) are immunoglobulins complimentary to polymorphic proteins that are different between the recipient and the graft donor, most DSAs are directed against donor human leukocyte antigen proteins, other DSAs such as angiotensin-II type-1 receptor antibodies, anti-glutathione S-transferase antibodies, and MHC-1-related chain A antibodies have been recognized. DSAs can be either preformed before LT or it can appear de novo after transplantation.
      • McCaughan J.A.
      • Tinckam K.J.
      Donor specific HLA antibodies & allograft injury: mechanisms, methods of detection, manifestations and management.
      ,
      • Del Bello A.
      • Congy-Jolivet N.
      • Danjoux M.
      • Muscari F.
      • Kamar N.
      Donor-specific antibodies and liver transplantation.
      DSAs are also associated with increased risk of graft rejection (Acute and chronic). One meta-analysis showed that compared to patients who do not develop de novo DSAs, patients who develop them have a higher risk of developing acute rejection (OR 6.43, CI 3.17–13.04 P < 0.001). This association was more pronounced in pediatric population (OR 10.2, CI 4.65–22.33; P < 0.001).
      • Beyzaei Z.
      • Geramizadeh B.
      • Bagheri Z.
      • Karimzadeh S.
      • Shojazadeh A.
      De novo donor specific antibody and long-term outcome after liver transplantation: a systematic review and meta-analysis.
      Banff work group updated criteria for diagnosing AMR include positive DSA, defined as mean fluorescence intensity (MFI) > 5000. MFI is a different metric for assessing DSA activity and was shown to have an association with clinical significance. MFI should be interpreted with caution as there is a lack of standard cut-off value and can be misleading if used as a single measure to evaluate AMR.
      • Demetris A.J.
      • Bellamy C.
      • Hübscher S.G.
      • et al.
      2016 comprehensive update of the Banff working group on liver allograft pathology: introduction of antibody-mediated rejection.
      ,
      • Tambur A.R.
      • Herrera N.D.
      • Haarberg K.M.
      • et al.
      Assessing antibody strength: comparison of MFI, C1q, and titer information.
      No studies evaluating biomarkers in patients receiving multiple organ transplantation were identified.

      Search for molecular diagnostics

      Biopsy with histopathology can help in differentiating the cause of liver damage or formulate a differential diagnosis, stage the damage, and guide the therapeutic strategies.
      • Naini B.V.M.D.
      • Lassman C.R.M.D.P.
      Liver transplant pathology.
      Biopsy can help differentiating the types of acute rejection (ATCMR, AMR, or chronic rejection) based on several criteria and stage each subtype.
      • Demetris A.J.
      • Bellamy C.
      • Hübscher S.G.
      • et al.
      2016 comprehensive update of the Banff working group on liver allograft pathology: introduction of antibody-mediated rejection.
      Despite being the SOC procedure, biopsy is far from ideal. Biopsy has two major limitations, inadequacy of liver biopsy sample, and variability of histopathological interpretation. Liver biopsy gives a representative sample of the entire liver tissue and Banff working group recommends a liver biopsy with a 16-gauge needle passing twice through the liver with more than 11 portal tracts to accurately represent the liver pathology. Reports showed that about 84% of biopsies fails to pass this threshold, and other reports showed that despite reaching this biopsy size, biopsy may miss the diagnosis suggesting that a sample of 11–16 portal tracts are necessary to adequately represent liver tissue.
      • Sanai F.M.
      • Keeffe E.B.
      Liver biopsy for histological assessment: the case against.
      Liver biopsy interpretation is subjective and intra-observer and inter-observer bias has been shown to affect interpretation even when using Banff criteria. A report of 102 liver biopsy samples showed 27% discordance between experienced pathologists’ assessment of the biopsies for acute rejection and assessment by the initial pathologist.
      • Coffin C.S.
      • Burak K.W.
      • Hart J.
      • Gao Z-h
      The impact of pathologist experience on liver transplant biopsy interpretation.
      Due to these shortcomings of liver biopsy and the fact that till now there are no reliable biomarkers for diagnosis of acute rejection, many groups started to search for ways to improve diagnostic yield of liver biopsy and avoid inter and intra-observer bias.

      Molecular microscopic diagnostic system

      The earliest application for microarrays was tested in the early 2000's in the setting of kidney transplantation.
      • Halloran P.F.
      • Einecke G.
      Microarrays and transcriptome analysis in renal transplantation.
      The use of microarrays analysis in the setting of LT was validated in INTERLIVER study. The study aimed to analyze rejection-associated transcripts using molecular microscopic diagnostic system (MMDx) in identifying injury and rejection in liver biopsies using machine learning models.
      • Madill-Thomsen K.
      • Abouljoud M.
      • Bhati C.
      • et al.
      The molecular diagnosis of rejection in liver transplant biopsies: first results of the INTERLIVER study.
      MMDx is a recent application of machine learning which uses microarrays to interpret human biopsies to improve the diagnostic value of liver biopsy. The validated computer models were trained to compare the histologic findings to database-stored biopsies. The models can provide data on rejection-related molecular changes. Additionally, it can provide an index which correlates with non-adherence or under immune-suppression.
      • Halloran P.F.
      • Madill-Thomsen K.S.
      The Molecular Microscope(®) Diagnostic System meets eminence-based medicine: a clinician's perspective.
      It can also predict survival of an organ with similar histological and molecular changes. MMDx shows higher reliability in kidney biopsies, and it showed diagnostic utility in both TCMR and AMR.
      • Madill-Thomsen K.S.
      • Wiggins R.C.
      • Eskandary F.
      • Böhmig G.A.
      • Halloran P.F.
      The effect of cortex/medulla proportions on molecular diagnoses in kidney transplant biopsies: rejection and injury can Be assessed in medulla.
      MMDX can correlate histological finding to liver chemistries which is helpful in assessing the severity of rejection and monitoring response to treatment. This led to more comprehensive reporting of biopsies which will open new horizons in the early management and prediction of acute rejection.
      • Madill-Thomsen K.
      • Abouljoud M.
      • Bhati C.
      • et al.
      The molecular diagnosis of rejection in liver transplant biopsies: first results of the INTERLIVER study.
      MMDX systems have also been studied to monitor dd cf-DNA in the serum to detect accurately diagnose acute rejection.
      • Uo Alberta
      Trifecta-Heart cfDNA-MMDx Study.
      ,
      • Uo Alberta
      Trifecta-Kidney cfDNA-MMDx Study.
      Multiple challenges are still facing MMDX. It depends on the databases of biopsies which in themselves may be imprecise. Earlier models showed discrepancy between histological and molecular patterns of rejections. Biopsies used to train the models cannot distinguish between inflammation due to rejection and inflammation due to non-rejection etiologies that creates inconsistencies in the system.
      • Randhawa P.S.
      The molecular microscope diagnostic system (MMDx) in transplantation: a pathologist's perspective.
      To overcome these challenges, multiple clinical trials are conducted in different solid organ transplantations to improve the system. Table 2 summarizes these trials.
      Table 2Clinical Trials Evaluating Molecular Microscopic Diagnostic System.
      Study nameStatusPrimary endpointLocalityNumber of biopsies
      INTERLIVER
      • Halloran P.
      Diagnostic and Therapeutic Applications of Microarrays in Liver Transplantation (INTERLIVER).
      RecruitingAssign molecular scores of ATCMR and AMR in liver transplant biopsiesMulticenter800 biopsies
      INTERHEART
      • Halloran P.
      Diagnostic and Therapeutic Applications of Microarrays in Heart Transplantation.
      RecruitingDiagnosis of ATCMR and AMR in heartMulticenter889 biopsies from 454 patients
      Trifecta-Kidney cfDNA-MMDx Study
      • Uo Alberta
      Trifecta-Kidney cfDNA-MMDx Study.
      RecruitingCompare DDcfDNA to MMDx, HLA, and histology in kidneyMulticenter700 biopsies and 2100 blood samples
      Trifecta-Heart cfDNA-MMDx Study
      • Uo Alberta
      Trifecta-Heart cfDNA-MMDx Study.
      RecruitingCompare DD-cfDNA to MMDx to calibrate diagnosis of ATCMR and AMR in heartMulticenter300 biopsies
      INTERCOMEX
      • Halloran P.
      Diagnostic and Therapeutic Applications in Microarrays in Organ Transplantation.
      RecruitingValidate the use of microarrays to reduce uncertainty in diagnosis of ATCMR and AMR in kidneyMulticenter1500 biopsies
      INTERLUNGEX
      • Halloran P.
      Diagnostic and Therapeutic Applications of Microarrays in Lung Transplantation.
      RecruitingDetect molecular identifiers in ATCMR and AMR in lung biopsiesMulticenter818 TBB and 657 3BMBs
      TBB; trans-bronchial biopsies, 3BMBs; third bifurcation mucosal endo-bronchial biopsies.

      Nano-string

      One of the drawbacks of the MMDX systems is their dependability on a different tissue preservation technique other than conventional formalin-fixed paraffin embedded preparation (FFPE) thus limiting the practical application of MMDX.
      • Madill-Thomsen K.
      • Perkowska-Ptasińska A.
      • Böhmig G.A.
      • et al.
      Discrepancy analysis comparing molecular and histology diagnoses in kidney transplant biopsies.
      Nano-string® is a highly multiplexed RNA sequencing system that can analyze up to 800 target genes at the same time, it can use the standard FFPE biopsies without the need for any special preparation which gives it the advantage of being able to be studied and compared retrospectively to the archived biopsies.
      • Adam B.
      • Afzali B.
      • Dominy K.M.
      • et al.
      Multiplexed color-coded probe-based gene expression assessment for clinical molecular diagnostics in formalin-fixed paraffin-embedded human renal allograft tissue.
      In a report evaluating liver biopsy from 133 pediatric LT, Nano-string® was used to analyze a model compromised of 194 genes to detect rejection in liver biopsies. The model was able to detect rejection (OR Log2 0.4 CI: Log2 0.2-Log2 0.6 P-value 0.05) and furthermore was able to give details on the underlying pathophysiological pathways of rejection.
      • Feng S.
      • Bucuvalas J.C.
      • Demetris A.J.
      • et al.
      Evidence of chronic allograft injury in liver biopsies from long-term pediatric recipients of liver transplants.
      Further studies are still needed to evaluate the different RNA models to determine the most significantly associated genes with liver rejection and then to test these models prospectively to determine clinical applicability.

      Banff human organ transplant gene panel

      Banff molecular diagnostic work group (MDWG) did a literature review to search for genes associated with rejection in solid organ transplantation and identified 760 genes with most association. Using the Nano-string® platform, MDWG created the Banff human organ transplant (B-HOT) gene panel, to be used as research tool aiming at reaching a simplified panel to detect and classify acute rejection.
      • Mengel M.
      • Loupy A.
      • Haas M.
      • et al.
      Banff 2019 Meeting Report: molecular diagnostics in solid organ transplantation–Consensus for the Banff Human Organ Transplant (B-HOT) gene panel and open source multicenter validation.
      A study evaluated B-HOT on a sample of renal transplant biopsies and found that this panel was a suitable surrogate for microarrays in the detection of genes associated with rejection. This is the first report to assess the efficacy of the new panel.
      • Smith R.N.
      In-silico performance, validation, and modeling of the Nanostring Banff Human Organ transplant gene panel using archival data from human kidney transplants.
      MDWG started a multicenter cooperation to validate B-HOT panel and explore the aspects of its clinical application to improve patient care.
      • Mengel M.
      • Loupy A.
      • Haas M.
      • et al.
      Banff 2019 Meeting Report: molecular diagnostics in solid organ transplantation–Consensus for the Banff Human Organ Transplant (B-HOT) gene panel and open source multicenter validation.
      Advances in molecular biology and artificial intelligence led to the development of multiple biomarkers and molecular diagnostic techniques, including micro-RNA, dd cfDNA, nano-string, and MMDX. We may need to engage expert pathologists to use both histology criteria and the molecular analyses and derive an integrated classification. Biomarkers and molecular diagnostics of liver rejection are still not ready for prime time and need more studies on multiple cohorts, including pediatric patients and simultaneous liver-kidney transplant recipients. Before any of them can be transferred from bench to bedside, we need prospective large scale multicenter studies to establish its role in the diagnosis of rejection and achieve the accuracy in predicting and preventing the occurrence of graft rejection. Only then we can achieve the optimization of immune-suppression, precision care with the prevention of long-term side-effects and graft loss, and improved survival with a better quality of life following transplantation.

      Author contributions

      All authors approved the final version of the manuscript.
      Ahmed El Sabagh: Conceptualization, Writing - Original Draft, Visualization. Islam B Mohamed: Writing - Original Draft. Fuad Zain Aloor: Visualization, Designing illustrations. Ahmed Abdelwahab: Writing - Review & Editing. Manal M. Hassan: Writing - Review & Editing. Prasun K Jalal: Conceptualization, Writing - Review & Editing, Supervision, Project administration.
      All authors: Critical revision of the manuscript.

      Conflicts of interest

      All authors have none to declare.

      Acknowledgments

      None.

      Funding

      Dora Roberts Foundation.

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