Abstract and Introduction
Abstract
Histologic diagnosis of T cell-mediated rejection is flawed by subjective assessments, nonspecific lesions and arbitrary rules. This study developed a molecular test for T cell-mediated rejection. We used microarray results from 403 kidney transplant biopsies to derive a classifier assigning T cell-mediated rejection scores to all biopsies, and compared these with histologic assessments. The score correlated with histologic lesions of T cell-mediated rejection (infiltrate, tubulitis). The accuracy of the classifier for the histology diagnoses was 89%. Very high and low molecular scores corresponded with unanimity among three pathologists on the presence or absence of T cell-mediated rejection, respectively. The molecular score had low sensitivity (50%) and positive predictive value (62%) for the histology diagnoses. However, histology showed similar disagreement between pathologists—only 45–56% sensitivity of one pathologist with diagnoses of T cell-mediated rejection by another. Discrepancies between molecular scores and histology were mostly when histology was ambiguous ("borderline") or unreliable, e.g. in cases with scarring or inflammation induced by tissue injury. Vasculitis (isolated v-lesion TCMR) was particularly discrepant, with most cases exhibiting low TCMR scores. We propose new rules to integrate molecular tests and histology into a precision diagnostic system that can reduce errors, ambiguity and interpathologist disagreement.
Introduction
T cell-mediated rejection (TCMR) remains important as a prototype for T cell-mediated inflammatory diseases, a target for immunosuppressive drug development, an end point for drug trials and a feature of the early transplant course, particularly with new treatment protocols. However, histologic diagnosis of TCMR by the Banff classification has serious limitations. It was derived as "acute rejection" before antibody-mediated rejection (ABMR) was defined, and relies on nonspecific lesions, arbitrary rules and subjective interpretations that have never been validated against an objective independent test. The principal rule for diagnosing TCMR requires semi quantitative assessment of two lesions, interstitial inflammation (i-score) and tubulitis (t-score), using arbitrary thresholds. Both lesions are observed in other conditions such as acute kidney injury (AKI) and primary renal diseases. In protocol biopsies, both correlate with previous injury, but not with rejection, because the inflammatory response to injury can produce these lesions. Many biopsies with inflammation and tubulitis below the thresholds are called 'borderline'. Furthermore, these lesions must be assessed in unscarred areas, rendering the diagnosis of TCMR impossible in biopsies with advanced atrophy scarring. The second rule for diagnosing TCMR relies on isolated v-lesions: intimal arteritis without sufficiently high i- and t-lesions (v > 0 and (i < 2 or t < 2)). This rule is suspect because v-lesions can result from ABMR and AKI, and will probably change at the next Banff meeting.
In addition, TCMR diagnoses are poorly reproducible within the rules. Furness et al. found that agreement among pathologists using the same rules was not only limited but resistant to improvement. These authors concluded that '…international variation in histologic grading is large, under recognized, difficult to improve, and almost certainly of major clinical relevance. Urgent steps are required to improve this area of clinical practice.
Given the absence of a reliable gold standard, molecular phenotyping presents an alternative approach which in other areas of medicine, particularly cancer, is in clinical use. Molecular studies of transplant biopsies using RTPCR and microarrays have shown promise, but none distinguished between TCMR and ABMR. The importance of this distinction is underscored by the discovery that most ABMR is missed by the Banff criteria requiring C4d staining. As a result, all earlier studies must now be reinterpreted: many biopsies previously called 'rejection' or TCMR are actually mixed TCMR/ABMR.
This study aimed to develop a molecular test for TCMR. We performed microarrays on 403 kidney transplant indication biopsies that had been assigned histology diagnoses incorporating both the Banff classification and new knowledge such as C4d-negative ABMR. The molecules that best distinguished TCMR from other diseases were used to develop classifier equations that assigned TCMR scores to every biopsy by cross-validation. We compared the scores to histologic lesions and diagnoses, and studied the discrepancies in relationship to known limitations of histology. The assumption was that TCMR can be detected by a molecular signal that will clarify biopsies that are ambiguous or unclassifiable by histology, e.g. scarred biopsies. This approach allowed us to estimate the potential error rate in the histologic diagnosis of TCMR, and to suggest how the molecular test can be used to improve biopsy assessment.