Introduction: The broad spectrum of morphologic changes in Banff Borderline category in renal transplants poses a potential problem for clinical decision making, therapy response monitoring, and development of new tests due to reference standard-related biases. The goal of our study was to test the applicability of novel tissue based molecular assays to fine tune the assessment of alloimmune activity in transplant kidney biopsies with low grade inflammation.
Material and Methods: Six months renal transplant protocol biopsies with Banff normal (N=4) and borderline changes (N=6) were included into this pilot study. Gene expression analysis (GEA) and in situ hybridization (ISH) were performed on formalin fixed paraffin embedded biopsies (FFPE). RNA was isolated from 3x10ɥm FFPE sections (Invitrogen), and for GEA nanoString nCounter Sprint platform was used with a customized panel of 30 genes relevant in alloimmune response. ISH assay was performed with a chromogenic detection method (ACDBio); additional immunohistochemistry (IHC) for CD45 was done, followed by whole-slide scanning and automated digital image analysis (Definiens). The ISH and IHC platforms were both validated in earlier studies. CXCL-9, -10, and LCK were selected for alloimmune monitoring, since all three are well known to be relevant in alloimmune response. ISH signal and CD45⁺ cells were quantified and expressed as spot count/1000 cells and cells/1 mm², respectively. Spearman’s rank correlation, Pearson’s correlation and Mann-Whitney test were used where applicable.
Result and Discussion: There was a strong correlation between the log2 transformed gene count measured by GEA and ISH spot count/1000cells for CXCL-9 (Spearman’s rho = .899), CXCL-10 (r = .841), and LCK (r=.772), p < 0.001 for all tests. The borderline cases showed a significantly higher expression level on the ISH platform for CXCL-10 (p=.031), and LCK (p=.042) and on the nanoString platform for CXCL-9 (p=.026), and LCK (p=.036), indicating a higher alloimmune activity at molecular level in borderline cases.  CD45⁺ cell count also revealed a significant difference between normal and borderline cases [111 cells/1 mm² (interquartile range, IQR = 199.5) and 421 cells/1 mm² (IQR = 461.3), p= .004)]. These data show that a multimodal, FFPE tissue-based gene expression and immune cell profiling approach can differentiate between normal cases and cases with low level inflammatory infiltrate.
Conclusion: The interpretation of borderline changes in transplant kidney biopsies remains challenging. Our quantitative ISH and immune cell profiling platform has the potential to go beyond the current gold standard of Banff classification and to monitor alloimmune responses in the kidney allografts at a molecular level. The described assays are currently being used in a clinical trial setting to evaluate the impact of a new treatment option in patients with borderline changes.