Analysis of proliferating cell nuclear antigen expression aids histological diagnosis and is predictive of progression of human cardiac allograft rejection

A quantitative immunohistological analysis was undertaken of 558 sequential paraffin-embedded and 22 snap-frozen endomyocardial biopsies (EMBs) from nine consecutive patients undergoing cardiac transplantation and followed for up to 1 year post-surgery. Serial monitoring was performed to assess whether 1) the phenotypic characteristics, 2) level of immune activation, or 3) expression of proliferation-associated antigens by intragraft leukocytes were useful in determining the grade of rejection or predicting further rejection episodes. Particular attention was given to those patients whose most recent EMBs showed grade 2 rejection, because these patients present a common problem in clinical management wherein a decision must be made as to whether or not to increase immunosuppression. Comparison of EMBs displaying various grades of rejection showed that whereas the absolute number of leukocytes (CD45), memory T cells (UCHL1/CD45RO), helper T cells (OPD4), and macrophages (Mac387) increased with increasing grade of rejection, the proportions of each subset remained similar. Cell proliferation was determined by labeling with monoclonal antibodies to proliferating cell nuclear antigen (cyclin) and Ki-67, and immune activation was assessed using an anti-interleukin-2 receptor (CD25) monoclonal antibody. The numbers of intragraft proliferating cell nuclear antigen-positive Ki-67+ or interleukin-2 receptor-positive cells were found to increase with increasing grades of rejection. Moreover, comparison of EMBs with equivalent histological features of rejection (grade 2) showed significantly (P < 0.0001) greater numbers of proliferating cell nuclear antigen-positive cells in EMBs preceding an episode of higher grade or persisting rejection versus EMB from patients whose rejection resolved, as seen on subsequent biopsy, without increased immunosuppression. These data suggest that the identification of proliferating or immunologically activated cells may aid in the histological diagnosis of clinical rejection and provide a valuable indicator predictive of likely further rejection episodes of increasing severity if grade 2 rejection is left untreated.