Investigational methods in the diagnosis of acute kidney allograft rejection

INTRODUCTION — For decades, routine monitoring of allograft function after kidney transplantation has relied upon the use of nonspecific markers, such as serum creatinine, glomerular filtration rate (GFR), and proteinuria. The definitive diagnosis of kidney allograft dysfunction still requires an invasive allograft biopsy, which remains the gold standard for the assessment of graft status. Given their low sensitivity, existing markers fail to detect subclinical changes, and pathologic processes are often recognized at a relatively advanced stage. "Protocol" or screening biopsies have been proposed in kidney transplant recipients to identify early pathologic changes that may be evident before graft dysfunction. However, a number of limitations reduce the utility of screening biopsies in clinical practice: Biopsies are invasive, and complications may occur; sampling errors may jeopardize their diagnostic utility; and the procedures are costly and labor intensive. (See "Overview of care of the adult kidney transplant recipient", section on 'Monitoring kidney allograft function'.)

Innovative biomarkers have been developed to circumvent the shortcomings of existing methods for monitoring allograft function. Although several such markers have been found to provide good diagnostic and/or prognostic utility, few, if any, are used in clinical practice, and there is insufficient evidence that their use will lead to meaningful improvements in graft outcome [1]. Additional barriers that limit the introduction of novel biomarkers into clinical practice include regulatory issues, generalizability, cost, ease of interpretation, and identification of patient populations who may benefit from more than standard-of-care surveillance.

This topic will review investigational methods in the diagnosis of acute rejection of the kidney allograft. The clinical features, diagnosis, treatment, and prognosis of kidney allograft rejection, and the evaluation and diagnosis of kidney allograft dysfunction, are discussed separately:

●(See "Kidney transplantation in adults: Clinical features and diagnosis of acute kidney allograft rejection".)

●(See "Kidney transplantation in adults: Treatment of acute T cell-mediated (cellular) rejection".)

●(See "Kidney transplantation in adults: Prevention and treatment of antibody-mediated rejection".)

●(See "Kidney transplantation in adults: Evaluation and diagnosis of acute kidney allograft dysfunction".)

LIMITATIONS OF CURRENTLY USED BIOMARKERS

Kidney allograft biopsy — Although imperfect, the kidney allograft biopsy remains the gold standard to identify the mechanisms of graft injury in kidney transplant recipients [2,3]. Assessment of the predictive performance of a biomarker (ie, sensitivity, specificity, and positive and negative predictive values) requires knowledge of the actual diagnosis. Because allograft biopsies are considered the gold standard, it is impossible to determine their sensitivity and specificity, despite known limitations of biopsy data. Specifically, the problem of sampling error likely results in biopsies that do not include tissue that is adversely affected by pathologic processes, thereby increasing the proportion of false-negative tests (ie, the absence of a histologic lesion is not proof of the absence of this lesion). However, no other diagnostic method can provide information about the true state of the allograft, and, therefore, the actual diagnostic performance of the allograft biopsy remains unknown. In addition, pathologists frequently vary in their interpretation of biopsy findings. (See "Overview of care of the adult kidney transplant recipient", section on 'Kidney biopsy'.)

Serum creatinine — Serum creatinine concentration is one of the primary markers used to monitor kidney allograft function in clinical practice. Early graft function, as determined by measurement of serum creatinine at one year posttransplant, has been shown to predict long-term graft survival [4]. However, at an individual level, a serum creatinine value is neither sensitive nor specific for graft injury [5]. Subclinical rejection develops in the absence of overt graft dysfunction. In addition, an increase in serum creatinine after transplantation may reflect a variety of underlying processes including acute tubular injury, immunosuppressive drug toxicity, obstructive nephropathy, and infection-associated nephropathy. (See "Overview of care of the adult kidney transplant recipient", section on 'Serum creatinine and GFR' and "Kidney transplantation in adults: Evaluation and diagnosis of acute kidney allograft dysfunction", section on 'Causes of acute allograft dysfunction'.)

Proteinuria — Proteinuria is routinely measured with serum creatinine to monitor graft function after kidney transplantation [6,7]. Several observational studies have shown that increased urine protein excretion posttransplant is an important and independent predictor of graft failure [8-13]. However, albuminuria and proteinuria are nonspecific markers of graft injury, and an association between proteinuria and specific pathologic processes in the kidney allograft has not been well described [8,9,14]. One study of 1518 kidney transplant recipients evaluated the diagnostic and prognostic performance of measuring proteinuria after transplantation, at the time of graft dysfunction (with clinically indicated biopsies), and during routine clinical follow-up (with protocol biopsies) [13]. After three months posttransplant, proteinuria >1 g/24 hours at the time of a clinically indicated biopsy had a high specificity (85 to 91 percent) but low sensitivity (21 to 48 percent) for the combination of transplant glomerulopathy, microcirculation inflammation, and/or de novo/recurrent glomerular disease. Thus, biomarkers that are more sensitive for detecting potentially treatable graft injuries are needed. (See "Overview of care of the adult kidney transplant recipient", section on 'Proteinuria'.)

DSA monitoring — High levels of preformed donor-specific antibodies (DSAs) at the time of transplantation are generally considered a contraindication for successful transplantation. Low levels of DSAs, which are not a contraindication to transplant, may subsequently lead to a wide spectrum of antibody-mediated graft injuries and, therefore, require individual risk assessment. While advances in screening methods have increased the sensitivity for detecting very low levels of DSAs, the specificity and positive predictive value (PPV) of DSA for the risk of developing antibody-mediated rejection (ABMR) have decreased. As an example, in one study of 402 deceased-donor kidney transplant recipients, the sensitivity, specificity, and PPV of a remote positive complement-dependent cytotoxicity crossmatch for detecting subsequent ABMR were 41, 97, and 54 percent [15]. By comparison, with the single-antigen (Luminex) bead assay, the sensitivity, specificity, and PPV of a preformed DSA on the peak serum were 91, 85, and 35 percent. (See "Kidney transplantation in adults: Clinical features and diagnosis of acute kidney allograft rejection" and "Kidney transplantation in adults: Prevention and treatment of antibody-mediated rejection", section on 'Predictors of outcome'.)

Additional evidence has suggested that the development of DSAs after transplantation (ie, de novo DSAs) is associated with decreased graft survival [16,17]. However, despite the widespread use of posttransplant monitoring for DSAs, the utility of this strategy to noninvasively diagnose ABMR or predict long-term graft outcomes has not been clearly evaluated. In one study of 281 indication biopsies (ie, biopsies performed in the setting of allograft dysfunction) in 244 kidney transplant recipients, the presence of DSA (both preexisting and de novo) at the time of allograft biopsy was associated with the diagnosis of ABMR (odds ratio [OR] 3.6 and 10.4 for mean fluorescence intensity [MFI] of 1000 to 3000 and >3000, respectively) [5]. However, many patients with DSAs did not have ABMR, suggesting that DSA should be considered as a risk factor for rather than diagnostic of ABMR. Another study reported an association between de novo DSAs and the histologic findings of peritubular capillaritis and C4d deposition on allograft biopsy, but the percentage of biopsies meeting the criteria for ABMR was not provided [16].

NONINVASIVE BIOMARKERS — The limitations of existing markers of kidney allograft dysfunction highlight the need for innovative, robust, and, ideally, noninvasive methods to predict and diagnose acute and chronic graft injury. The development of "omics" methods (eg, genomics, transcriptomics, proteomics, metabolomics) in the field of transplantation has paved the way for the development of several candidate biomarkers [18-24]. The following discussion will highlight some of these approaches to noninvasive or minimally invasive monitoring of the kidney transplant.

Functional cell-based immune monitoring — Alloreactive memory T cells are central mediators of kidney allograft rejection, and monitoring the activity of these cells may help to identify transplant recipients who are at risk for acute rejection (see "Transplantation immunobiology", section on 'Effector mechanisms associated with allograft rejection'). The interferon (IFN)-gamma enzyme-linked immunospot assay (ELISPOT), which measures IFN-gamma secretion by recipient T cells in response to donor antigens, is one test that has been used to assess antidonor T cell alloreactivity in vitro [25]. Several studies have found that pretransplant T cell alloreactivity, as determined by the IFN-gamma ELISPOT, may be associated with an increased risk of posttransplant acute rejection and lower posttransplant graft function [25-30]. However, in one multicenter observational study of 176 kidney transplant recipients, a correlation between pretransplant ELISPOT reactivity and acute rejection or posttransplant graft function was only observed in patients who did not receive induction therapy with rabbit antithymocyte globulin (rATG)-Thymoglobulin [31]. One study found that the persistence of donor-specific T cell alloreactivity at 6 months after transplantation, under conditions of potent immunosuppression, was associated with subclinical T cell-mediated rejection (TCMR) and worse allograft function at 12 months posttransplant [32].

Although assays such as the IFN-gamma ELISPOT may be helpful in monitoring T cell alloreactivity and memory B cells, wider application may be limited by their laborious, time-consuming, and impractical use in clinical practice.

Molecular blood biomarkers — Several studies have identified potential biomarkers in peripheral blood that may identify patients with acute kidney allograft rejection [33-42], including subclinical acute rejection [43,44]. The following discussion will focus on important advances in the field of peripheral blood messenger ribonucleic acid (mRNA) and donor-derived cell-free DNA (dd-cfDNA) analysis and the diagnosis and prediction of acute rejection.

Kidney Solid Organ Response Test (kSORT) — The Kidney Solid Organ Response Test (kSORT) is a microarray-based assay that was developed to detect patients at high risk for acute rejection [40]. The test, performed on a peripheral blood sample, employs quantitative polymerase chain reaction (PCR) to measure the relative mRNA expression levels of 17 genes known to be associated with acute rejection or leukocyte trafficking. A correlation-based algorithm was then used to generate risk scores and classify patients into high, low, or indeterminate risk of acute rejection. The utility of the kSORT assay was initially evaluated in a large multicenter study of 436 adult and pediatric kidney transplant recipients (Assessment of Acute Rejection in Renal Transplantation [AART]) with paired peripheral blood samples and kidney allograft biopsies (performed for allograft dysfunction or as part of a clinical protocol) using mainly a case-control study design of selected patients [40]. The kSORT assay was able to predict patients at high risk of acute rejection (either TCMR or antibody-mediated rejection [ABMR]) with a sensitivity and specificity of 92 and 93 percent, respectively. In addition, kSORT was able to identify 12 of 16 biopsies with subclinical rejection and could predict clinical rejection in more than 60 percent of samples collected within three months before the diagnosis of biopsy-confirmed acute rejection. However, the test was unable to distinguish between acute TCMR and ABMR.

A subsequent study evaluated the use of kSORT and the IFN-gamma ELISPOT assays (see 'Functional cell-based immune monitoring' above) in predicting subclinical acute rejection [41]. In a cohort of 75 kidney transplant recipients, combined use of both the kSORT and ELISPOT assays improved the predictive probability for subclinical acute rejection and the ability to distinguish between subclinical TCMR and ABMR. However, a subsequent large, multicenter study of 1134 patients using blood samples obtained under real-world clinical conditions was unable to validate the utility of the kSORT assay for the detection of acute rejection in the first year after transplantation [45].

TruGraf v1 assay — The TruGraf assay is a DNA microarray-based gene expression blood test that was developed as an alternative to surveillance biopsies to rule out subclinical rejection in patients with stable graft function [46]. All aspects of discovery and external validation of the TruGraf test were performed on blood samples paired with protocol biopsies from prevalent cohorts. How the test performs in patients with graft dysfunction has not been assessed and remains to be studied. One study found that combining the TruGraf assay with dd-cfDNA, another noninvasive biomarker used for the diagnosis of acute rejection, improved the detection of subclinical rejection [47]. (See "Kidney transplantation in adults: Clinical features and diagnosis of acute kidney allograft rejection".)

Other peripheral blood assays — Other assays have been developed to distinguish patients with and without acute rejection based upon the analysis of peripheral blood samples. As examples:

●In one multicenter study, global gene expression profiling was performed on peripheral blood samples from 148 kidney transplant recipients with normal histology and stable graft function (46 patients), biopsy-proven acute rejection (63 patients), or acute allograft dysfunction without rejection (39 patients) [42]. Analysis of these data identified 200 probe sets that distinguished patients with acute rejection from those without rejection with a sensitivity of 82 to 100 percent and specificity of 76 to 95 percent. However, the study was limited by the lack of prospective validation and absence of patients with ABMR. A prospective trial evaluating the diagnostic and prognostic utility of this assay in 300 kidney transplant recipients is in progress.

●One study found that a 17-gene signature in peripheral blood could identify patients with subclinical and borderline TCMR at three months posttransplant [44]. Such patients had a higher risk of subsequent clinical acute rejection at 12 and 24 months, faster decline in graft function, and decreased graft survival compared with those without subclinical or borderline TCMR at three months posttransplant.

●A multicenter study from the European BIOMARGIN consortium developed and validated an eight-gene expression assay for the diagnosis of ABMR [48]. This assay was able to differentiate between cases with and without ABMR with good diagnostic accuracy (area under the receiver operative characteristic curve [ROC AUC] 79.9 percent, 95% CI 72.6-87.2), both at the time of stable graft function and at the time of graft dysfunction. Adding a clinical model for the diagnosis of ABMR to the eight-gene assay further improved diagnostic accuracy.

●Quantification of microRNAs (miRNAs) in peripheral blood may represent a noninvasive biomarker for evaluation of allograft status. In one study, combined measurement of five miRNAs (miR-15B, miR-16, miR-103A, miR-106A, and miR-107) in peripheral blood samples enhanced the sensitivity and specificity for the diagnosis of severe T cell-mediated vascular rejection [37].

Urine biomarkers — Several urinary biomarkers, including mRNAs, miRNAs, and proteins and peptides, have been proposed as noninvasive biomarkers for the diagnosis and prognosis of acute rejection.

Urine messenger RNAs — Acute TCMR is characterized by interstitial inflammation and tubulitis with graft-infiltrating cells entering into the tubular space. This has led some investigators to hypothesize that the kidney allograft may function as an "in vivo flow cytometer" that sorts cells involved in rejection into the urine and that profiling of the urinary cell mRNA could serve as a noninvasive method to diagnose acute TCMR [19,49]. Single-center studies of urinary cell mRNA profiling identified several potential mRNAs (eg, perforin, granzyme B, IFN-inducible protein-10 [IP-10], CD3), the levels of which predicted the diagnosis of acute TCMR [50-53]. The most promising of these markers were prospectively evaluated in a large, multicenter study (Clinical Trials in Organ Transplantation 04 [CTOT4]) of 485 kidney transplant recipients [54]. A three-gene signature of CD3-epsilon mRNA, IP-10 mRNA, and 18S ribosomal RNA (rRNA) was able to distinguish between kidney biopsy specimens showing acute TCMR and those without rejection with a sensitivity of 71 to 79 percent and specificity of 72 to 78 percent. In addition, the molecular signature could distinguish acute TCMR from borderline rejection, acute ABMR, or chronic allograft nephropathy with a sensitivity and specificity of 79 and 71 percent, respectively. Evaluation of the three-gene signature in repeated urine samples over time suggested that this test could also predict the development of acute TCMR weeks before the onset of graft dysfunction. Global transcriptional analysis of urinary cell samples with RNA sequencing has also identified enriched gene signatures and pathways associated with TCMR and ABMR [55].

While these results appear promising, the potential for extensive degradation of mRNAs in urine is one important limitation of this assay. In the CTOT4 study cited above, for example, only 83 percent of urine samples passed quality control standards [54]. However, despite this limitation, a multicenter evaluation of a standardized protocol for urinary cell mRNA profiling demonstrated a reasonably good concordance between laboratories, confirming the potential of this technique in real-life clinical settings [56].

Urine microRNAs — Quantification of miRNA in urine samples has also emerged as an alternative method to assess allograft status [57]. The following studies illustrate the range of findings:

●In an initial study that profiled urinary miRNAs of stable transplant patients and those with acute TCMR, miR-10a, miR-10b, and miR-210 were strongly deregulated in the urine of patients with acute TCMR [58]. Low miR-210 levels were also associated with a greater decline in glomerular filtration rate (GFR) at one year posttransplant.

●One study evaluated miRNA expression profiles in allograft biopsy tissue and paired urine samples of transplant recipients with chronic allograft dysfunction and interstitial fibrosis/tubular atrophy (IF/TA) and those with normal allografts [59]. Fifty-six miRNAs were identified as differentially expressed in biopsy samples with IF/TA; five of these (miR-142-3p, miR-204, miR-107, miR-211, and miR-32) were confirmed using an independent set of samples. Differential expression of miR-142-3p, miR-204, and miR-211 in the urine of patients with IF/TA was also observed. Findings of a second study suggested that the combination of miRNA profiling of biopsy and urine samples could be used to monitor graft function and predict progression to chronic allograft dysfunction [60].

These preliminary findings will need to be validated in independent and unselected populations to assess the diagnostic and/or prognostic accuracy as well as the clinical utility of urine miRNAs. In addition, as the aforementioned studies quantified miRNAs in whole urine, urine cell pellets, or cell-free urine supernatant, the ideal medium to be used for urine miRNA profiling is uncertain.

Urine proteins — A number of urinary immune-related proteins have been identified as biomarkers of acute rejection of the kidney allograft [61]. As examples, elevated urinary levels of chemokine (C-X-C motif) ligands 9 and 10 (CXCL9 and CXCL10) have been associated with acute TCMR [61-68]. In a multicenter observational study of 280 adult and pediatric kidney transplant recipients (Clinical Trial in Organ Transplant [CTOT1]) that evaluated multiple urinary proteins and mRNAs as biomarkers of acute rejection, elevated urinary CXCL9 levels had a PPV and NPV of 68 and 92 percent, respectively, for diagnosing Banff ≥1A TCMR [62]. Urinary levels of CXCL10 are also elevated in patients with ABMR [5]. In a study of 244 kidney transplant patients, elevated urinary CXCL10 levels had a PPV and NPV of 55 and 86 percent for the diagnosis of ABMR. However, increased urinary CXCL9 and CXCL10 levels have also been detected in patients with BK polyomavirus (BKPyV) infection [66], suggesting that these chemokines may represent markers of allograft inflammation rather than specific diagnostic markers of acute rejection. One study in kidney transplant recipients found that urinary CXCL10 levels increased with BKPyV viremia but not with isolated viruria [69].

Some studies have suggested that measuring urinary chemokines may have prognostic value in kidney transplant recipients. One study, for example, found that urinary concentration of chemokine (C-C motif) ligand 2 (CCL2, also known as monocyte chemoattractant protein 1 [MPC1]) at six months posttransplant was a predictor of severe IF/TA and graft dysfunction at two years posttransplant [70,71]. In the CTOT1 study cited above, patients with stable allografts and low urinary CXCL9 levels at 6 months posttransplant were least likely to subsequently develop TCMR or a reduction in estimated GFR (eGFR) between 6 and 24 months [62]. Similarly, in another study, low urinary CXCL10 levels at one and three months posttransplant were associated with improved rejection-free allograft survival at one year posttransplant [72]. Urinary CXCL10 at three months independently predicted the subsequent development of clinical acute rejection with a PPV and NPV of 13 and 97 percent, respectively. Although the PPV was low, the high NPV suggested that urinary CXCL10 levels could serve to identify patients at low risk of rejection who might be optimal candidates for immunosuppressive drug weaning.

Urine proteomics/peptidomics — High-throughput methods have been used to identify proteomic and peptidomic signatures of acute rejection in urine and blood samples. Several studies have suggested that proteomic or peptidomic signatures can be used to noninvasively diagnose acute rejection with high sensitivity and specificity [73]. Proteins and peptides differentially expressed in patients with acute rejection have been identified, including fragments of collagens, beta-2-microglobulin, alpha-1-antichymotrypsin, and uromodulin. In addition, the high-throughput approach has enabled pathway-enrichment analysis that has uncovered key pathogenic processes, such as major histocompatibility complex (MHC) antigen presentation; IFN-gamma and integrin signaling; complement activation; platelet function; and keratin sulfate, glycosaminoglycan, and collagen metabolism.

While these findings support the applicability of proteomic approaches in the diagnosis of acute rejection, additional points need to be addressed. As an example, fewer studies have performed proteomic profiling of other histopathologic entities such as subclinical rejection [74-76], ABMR [77-81], mixed rejection [82], and chronic rejection phenotypes [78,79,83], which have only more recently been specifically defined in the Banff classification system. In addition, the lack of large-scale studies prevents the assessment of confounding factors, such as urinary tract infection, cytomegalovirus infection, or BKV nephropathy, or of superimposed histologic lesions such as acute tubular injury [84] that may associate with distinct proteomic signatures. Thus, longitudinal evaluation of these new assays should be performed in unselected, consecutively recruited patients [73]. Another aspect that has not been addressed is the potential use of proteomic approaches as prognostic biomarkers (ie, to anticipate rejection or graft outcome) or as predictive biomarkers (ie, to assess response to antirejection treatment). Finally, implementing proteomic markers into widespread clinical application will require standardization of the preanalytical procedures for sample preparation and development of simplified test systems.

Imaging methods — In addition to the biomarkers discussed above, medical imaging methods have also been developed as noninvasive approaches to diagnosing graft dysfunction and assessing graft status. As examples:

●In one study of 31 kidney transplant recipients with suspected acute rejection, 18-fluoro-2-deoxyglucose (FDG) positron emission tomography coupled with computed tomography (PET/CT) was able to distinguish nonrejection from acute rejection [85], suggesting that this test might be used to identify patients for whom an allograft biopsy could be avoided.

●Assessment of high-energy phosphates metabolism using 31-phosphorus magnetic resonance spectroscopy (MRS) has been used to evaluate kidney allograft status. One study of kidney transplant recipients with type 1 diabetes found that kidney-pancreas transplantation, compared with kidney-alone transplantation, was associated with a higher beta-adenosine triphosphate (ATP)/inorganic phosphorus ratio (a marker of graft energy status), suggesting that restoration of beta cell function positively affects kidney graft metabolism [86].

INVASIVE BIOMARKERS — Although there are several advantages to the use of noninvasive biomarkers, invasive diagnostic tests are frequently necessary to confirm the diagnosis when noninvasive testing reveals a high probability of active disease [24]. Invasive biomarkers may also be required to detect subclinical disease when sensitive noninvasive biomarkers are not available.

The molecular microscope — The "molecular microscope diagnostic system" (MMDx) is a microarray-based system that analyzes messenger RNA (mRNA) expression patterns in transplant biopsy tissue to predict the diagnosis of acute T cell-mediated rejection (TCMR) or antibody-mediated rejection (ABMR) [87]. This technology was developed with the hypothesis that the diagnosis of specific disease states can be established on the basis of mRNA levels in the biopsy sample [87,88].

The general procedure for the MMDx is as follows [87,89]:

●A kidney allograft biopsy sample (obtained with an 18 gauge or larger biopsy needle) is preserved in a tissue storage reagent that stabilizes RNA, then shipped by courier to a central processing laboratory that performs MMDx.

●RNA is extracted from the biopsy sample, and a microarray analysis is performed to measure mRNA levels.

●Predefined computer algorithms (known as classifiers) are used to generate a report (typically within 29 hours) that compares the results of the microarray analysis with a reference set of biopsy samples with established diagnoses. The report provides molecular scores that reflect the probability that the biopsy sample has histologic features of TCMR [90], ABMR [91], rejection of any type [92], acute kidney injury (AKI) [93], or interstitial fibrosis/tubular atrophy (IF/TA) [94].

The development of this assay was based upon the findings of several studies in kidney transplant patients that characterized and validated the molecular phenotypes ("molecular landscapes") for TCMR, ABMR, AKI, and IF/TA [87,90-100]. The following studies illustrate the range of findings:

●In one study, microarray results from 403 kidney transplant biopsies (35 with a diagnosis of TCMR) were used to derive a classifier that assigned TCMR scores to all biopsies; scores were then compared with the histologic diagnosis of the biopsies [90]. Transcripts that distinguished biopsies with TCMR from other transplant biopsies were primarily associated with effector T cell and macrophage infiltration and activation. Molecular scores correlated with histologic lesions of TCMR (tubulitis and interstitial infiltration) with an accuracy of 89 percent. The positive predictive value (PPV) and negative predictive value (NPV) of a high TCMR molecular score (≥0.1) for predicting the histologic diagnosis of TCMR were 62 and 92 percent, respectively.

●In a parallel study from the same group, microarray results from the same 403 kidney transplant biopsies (56 with a histologic/donor-specific antibody [DSA] diagnosis of ABMR) were used to derive a classifier that assigned ABMR scores to all biopsies [91]. Most of the transcripts that distinguished biopsies with ABMR from other transplant biopsies were expressed in natural killer (NK) cells or endothelial cells or were interferon (IFN)-gamma inducible. A positive ABMR molecular score (≥0.2) had a PPV and NPV of 64 and 91 percent, respectively, for predicting the histologic/DSA diagnosis of ABMR. In addition, ABMR molecular scores were strongly associated with DSA positivity and were an independent predictor of graft failure.

●The performance of the TCMR and ABMR molecular scoring systems was prospectively evaluated in a multicenter study (Integrated Diagnostic System in the International Collaborative Microarray [INTERCOM]) of 300 kidney transplant biopsies from 264 patients [95,96]. Histologic diagnoses of TCMR or ABMR (including C4d-positive ABMR, C4d-negative ABMR, and mixed ABMR and TCMR rejection) were present in 11 and 15 percent of biopsies, respectively. A TCMR molecular score of ≥0.1 predicted the histologic diagnosis of TCMR (including borderline rejection) with a PPV and NPV of 49 and 94 percent, respectively. Similarly, an ABMR molecular score of ≥0.2 predicted the diagnosis of ABMR or mixed rejection with PPV and NPV of 50 and 94 percent, respectively. There was no significant difference in the ABMR score between biopsies with C4d-positive ABMR and those with C4d-negative ABMR. A positive ABMR score in late biopsies (performed more than one year posttransplant) was significantly associated with death-censored graft loss at three years (hazard ratio [HR] 2.93, 95% CI 1.97-4.36); no association between the TCMR score and death-censored graft loss was observed.

Collectively, the results of these studies demonstrate that molecular phenotyping of kidney transplant biopsies is capable of discriminating between rejection and nonrejection at least as well as, if not better than, histologic evaluation and DSA alone. Discrepancies between molecular and histologic diagnoses, as evidenced by the relatively low PPVs in the studies discussed above, reflect a combination of issues including histologic errors or ambiguity, MMDx errors, and sampling differences (ie, different biopsy cores assessed by histology and MMDx) [87]. Microarray analysis of kidney allograft biopsy tissue has also been used to predict the risk of fibrosis and allograft loss [101].

Alternative methods have been developed and applied for transcriptomic analysis of the kidney allograft, including RNA sequencing [102] and the NanoString technology [103].

SUMMARY

●Limitations of existing biomarkers – Routine monitoring of allograft function after kidney transplantation has relied upon the use of nonspecific markers, such as serum creatinine, glomerular filtration rate (GFR), proteinuria, and donor-specific antibodies (DSAs). The definitive diagnosis of kidney allograft dysfunction still requires an invasive allograft biopsy, which remains the gold standard for the assessment of graft status. Given their low sensitivity, existing markers fail to detect subclinical changes, and pathologic processes are often recognized at a relatively advanced stage. (See 'Limitations of currently used biomarkers' above.)

●Investigational biomarkers – Innovative strategies have been developed to circumvent the limitations of existing biomarkers.

•Several noninvasive monitoring tools have been proposed that use easily accessible biologic fluids such as urine and blood, allowing frequent and sequential assessments of a recipient's immune status. These include functional cell-based assays and the evaluation of molecular expression, at the messenger RNA (mRNA) or protein level, on a wide spectrum of platforms. (See 'Noninvasive biomarkers' above.)

•Molecular technologies, including the molecular microscope diagnostic system (MMDx), have been developed as a refinement of the histologic evaluation of the allograft biopsy. (See 'The molecular microscope' above.)

The translation and validation of exploratory findings and their implementation into standard clinical practice remain challenging. Dedicated, prospective, interventional trials are required to demonstrate that the use of these biomarkers improves patient or transplant outcomes.

●Barriers to use in clinical practice – Significant barriers exist that limit the introduction of novel biomarkers into clinical practice. These include regulatory issues, generalizability, cost, ease of interpretation, and also identification of patient populations who may benefit from more than standard-of-care surveillance.