Addition of kSORT Assay in the Follow-up of Patients After Kidney Transplantation

I. Background Kidney transplantation is the treatment of choice in patients with end stage renal disease in need of function replacement therapy, and in whom there is no absolute contraindication to the procedure. The current figures reported in the USA on post-transplant graft survival at one year, are 95% in cases of living donor (LD) recipients and 90% in deceased donor (DD) recipients3; the mean calculated survival of these grafts has increased and is currently 14 years in LD and 11 years in DD recipients. The fact that graft survival hinges on multiple immune and non-immune factors is well recognized; however, the main cause of graft loss is solidly linked to its rejection, with evidence of involvement of the immune humoral response in most cases (specific antibodies against HLA antigens).

The main cause of acute graft dysfunction is acute rejection (AR). Several studies have associated episodes of this entity with chronic structural injury and subsequent functional impairment. Thus, timely diagnostic certainty is pivotal so as to optimize immunosuppressive therapy and preserve graft function. To date, the only tool available to confirm AR is graft biopsy and its established histological diagnostic criteria8. However, this diagnostic tool has limitations resulting mainly from sampling errors and costs. Furthermore, about 10% of patients with normal graft function have evidence of AR in protocol biopsies, so centers that do not use this follow-up modality in kidney transplant recipients (KTR) are unable to document this immune phenomenon. Ideally, an AR non-invasive, diagnostic tool would be of utmost use in KTR follow-up since it would preclude the need for graft biopsies and efficiently support the management of immune suppressors. Therefore, the systematic performance of a highly specific and sensitive assay reflecting AR, would allow for the timely detection of this immune phenomenon compared to the current standard (biopsy). Moreover, the systematic use of a tool with these characteristics would allow stratification of the immunological risk as well as proactive adjustment of immunosuppressor drug dosages, potentially limiting chronic injury and improving the graft's survival.

The background to the proposed study is based on the extensive line of research conducted by Dr. Sarwal et al. They have recently published results10 on monitored pediatric kidney transplant recipients. Biomarkers of gene panels in peripheral blood detected by microarray were discovered in a single institution and subsequently validated by quantitative-PCR in 12 pediatric kidney transplant programs. A total of 367 individual blood samples, each paired to a graft biopsy for blinded centralized classification, were analyzed (115 with acute rejection, 180 with stable kidney graft function, and 72 with other causes of graft injury). Among the genes differentially expressed in microarrays, quantitative-PCR analysis of a set of 5 genes (DUSP1, PBEF1, PSEN1, MAPK9 and NLTR) classified acute rejection with great precision. The 5 gene model has a sensitivity of 91%, specificity of 94%, positive predictive value of 83%, negative predictive value of 97% and a 92% precision when differentiating acute rejection samples from other phenotypes (stable and no acute rejection, no acute rejection/ non-stable). Interestingly, 8/12 samples from patients with borderline rejection on biopsy, were classified as acute rejection with the 5 gene model. The frequent prediction of the phenotype in borderline rejection biopsies suggests that pre-clinical injury in acute rejection could also be identified by quantitative-PCR in peripheral blood samples, thus allowing earlier treatment of these patients.10 The fact that this model predicts acute rejection in both cellular and humoral immune injury modalities, is of utmost relevance. Moreover, these genes in peripheral blood, strongly linked to graft rejection, do not correlate (are independent) with many demographic, clinical, treatment modalities and bacterial/viral infection parameters.

The 5 genes are centered on leukocyte trafficking and T/B cell activation and are mostly expressed in monocytes, activated in the peripheral circulation; they reflect injury response mechanisms to cellular oxidative stress (DUSP1), apoptosis (MAPK9), IL2-dependent cytolytic gene activation (NKTR), increased cell adhesion via the e-cadherin/catenin complex (PSEN1) and smooth muscle vascular cell injury (PBEF1).

The authors pointed out that the study was conducted in a pediatric and young adult population, in whom rejection may be more aggressive due to the disparity between the adult transplanted organ and the pediatric recipient, or as a result of poor treatment adherence in adolescent recipients, leading to a stronger immune response. Thus, the 5-gene model would require further evaluation in order to determine its diagnostic potential of acute rejection in patients of all ages, in larger patient cohorts and in association with different immunosuppressant regimes.