Precision Sub-phenotyping of Histologically Stable Kidney Transplants by a Composite Molecular and Cellular Instability Score

Acute Rejection (AR) is the main cause of the graft dysfunction and premature graft loss, and diagnosis of rejection before advanced histological injury is crucial to salvage graft function. However, recent molecular studies have highlighted the unrecognized burden of sub-clinical graft rejection when graft function is preserved, and a dichotomy exists, of a histologically normal biopsy with molecular signatures of AR. Conversely, significant variation also exists in the definition of a stable allograft, defined as a transplant with absence of clinical AR, with absence of histological inflammation, though published studies have highlighted that some of these stable samples will not have stable immune quiescence, as they may be molecularly similar to AR. Thus, refining the definition of a stable allograft as one that is clinically, histologically and molecularly quiescent is critical, as the inclusion of stable allografts in mechanistic and clinical studies are vital to provide a normal, non-injured comparative group for all interrogative studies on understanding allograft injury. With this goal in mind, we analyzed publicly available transcriptional data across 4,845 human kidney tissue samples from 38 Gene Expression Omnibus (GEO) datasets, inclusive of 510 allograft biopsy samples with AR, 1,154 renal allograft biopsies classified in each dataset as histological stable (hSTA), and 609 normal kidney (donor) samples. By applying a machine learning model, a substantive number of hSTA samples were found to be molecularly similar to AR (mAR); these have been reclassified in this study as clinical and histological stable samples with transcriptional signatures overlapping with AR (hSTA/mAR), with the predominant expression of a subset of 6 genes (KLF4, CENPJ, KLF2, PPP1R15A, FOSB, TNFAIP3). To understand the cellular sources of these molecular signals, we utilized xCell, a cell type enrichment tool and interrogated 64 specific cell types to identify 5 (CD4+ Tcm, CD4+ Tem, CD8+ Tem, NK cells, and Th1 cells) that were also highly predictive for classification of the AR phenotype in these studies. A combined gene and cell-type specific InstaScore (AUC 0.99) was developed using gene and cell subtype data to re-phenotype all hSTA allografts. This clearly defined two disparate hSTA biopsies: those that are both histologically and molecularly quiescent (hSTA/mSTA) or those that are histologically quiescent but molecularly similar to AR (hSTA/mAR). The clinical utility of the Instability Score was subsequently assessed by independent validation on a serial set of post-transplant hSTA biopsies, where strong significant correlation was observed between the score on 6 month post-transplant hSTA graft biopsies, where hSTA/mAR samples had a significant change in graft function and graft loss at 5 year follow-up. In conclusion, our computational approach of precision sub-phenotyping of hSTA allografts by the InstaScore identifies discrepancies in the current recognition of a stable allograft by histology alone. Precision molecular sub-typing of the hSTA allograft into the hSTA/mSTA group is an important deliverable for selection of "true" STA samples for mechanistic studies, and into the hSTA/mAR group, for accurate prediction of subsequent patient clinical outcomes, and real time treatment stratification for hSTA/mAR allografts to positively impact long-term graft survival.