Soluble Aminopeptidase N (CD13) Is a Diagnostic Biomarker of Late-Onset Chronic Graft Vs. Host Disease in Adults

Background:Chronic graft vs. host disease (cGVHD) is a major cause of morbidity and mortality after allogeneic HSCT. As an insidious onset and heterogeneous presentation renders this disease difficult to diagnose, there is a need for validated diagnostic biomarkers. Previously, soluble aminopeptidase N (CD13) was identified in a pediatric study as a biomarker for early onset cGVHD (diagnosed 3-9 months post transplant). Aminopeptidase N is a protease involved in immunoregulation on several levels; functions include attraction of T cells, antigen presentation, facilitation of adhesion and phagocytosis. Although it is integrated in the membrane of several cell types it can also be cleaved into soluble aminopeptidase N. In this study, we tested the potential plasma biomarker soluble aminopeptidase N in an adult population of late onset cGVHD patients using both a targeted method (enzymatic assay) as well as a non-targeted approach (proteomics).

Methods: Samples used in this study were frozen, EDTA-treated plasma samples derived from a single institution participating in the Chronic GVHD Consortium and consisted of 17 cases and 21 time-matched controls, all from adult patients. Cases were within 1 month of diagnosis of late-onset cGVHD (onset >9 months post transplant). Time posttransplant for cases vs. controls was 12 (9.2-26.8) vs. 11.9 (5.3-13.5) months, respectively. Other potential clinical variables included age at sample collection, gender, graft source, donor type, conditioning intensity, prior acute grade II-IV GVHD, and months from sample collection. Aminopeptidase N activity was determined by cleavage of L-leucine-p-nitroaniline; quantitative proteomic analyses were done with iTRAQ.

Results and Conclusions: Plasma from cGVHD patients had significantly higher mean levels of aminopeptidase N enzyme activity than did plasma from control patients (0.30 vs. 0.18 mU/ml,  respectively p=0.0008). Proteomic analyses using the same samples revealed that this difference was not restricted to activity; aminopeptidase N showed the most significant difference in protein levels corresponding to presence or absence of cGVHD of all the proteins identified. Relative amounts of soluble aminopeptidase N were 1.44 vs. 0.9, in cases and controls, respectively (p=0.0042). This study supports soluble aminopeptidase N as a potential diagnostic biomarker in adult GVHD. These results will be validated in a larger population which also includes early onset cGVHD.