Prospective Evaluation of Graft-Versus-Host Disease-Specific Proteomics Pattern in Patients after Allogeneic Stem Cell Transplantation.

We have recently described and published a proteomic pattern specific for the early diagnosis of acute graft versus host disease (aGvHD), based on the application of capillary electrophoresis (CE) and mass spectrometry (MS). Here we report the application of proteome based GvHD-patterns to prospectively collected samples from 86 patients (45 AML, 14 ALL, 8 MM, 5 CLL, 5 SAA, 3 MDS, 3CML, 3 NHL). Fifty three patients were transplanted from matched unrelated donors (MUD), 29 received stem cells from matched related donors (MRD), 3 from haplo-identical donors and 1 was transplanted from a syngeneic sibling. GvHD prophylaxis was methotrexat or mycophenolate and cyclosporin A in the majority of the patients. The follow up is currently between 60 and 549 days after allogeneic HSCT. Urine samples were collected on ice prior conditioning, once a week and monthly after day +60. To avoid degradation of the proteins/peptides the samples were frozen as soon as possible. After thawing and removal of confounding substances, like salts, lipids and of all molecules larger than 30 kDa, the samples were loaded onto the CE, separated according to their charge and, after ionization, directly analyzed in an electrospray ionization time of flight (ESI-TOF) -MS. Between 500 and 2500 peptides and proteins were detected in individual samples. All data generated are stored in a Microsoft MS database. Reproducibility of these analyses is greater 98%. The polypeptide patterns specific for the early detection of acute GvHD were applied to the data generated prospectively and the outcome of these analyses was compared to the clinical diagnosis of aGvHD, sepsis and CMV-reactivation. Forty patients were diagnosed with aGvHD in the current evaluation period, 18 had aGvHD ≥ II. From 461 samples, 68 gave a score with the aGvHD pattern, 15 were false positive, whereas 5 scored false negative. The sensitivity of the aGvHD pattern is more than 90%, the specificity is about 96%. Thus the application of the aGvHD pattern for early recognition of acute GvHD is very useful for predicting the development of aGvHD. In addition, we are currently evaluating samples of patients with chronic GvHD (cGvHD). Seven patients in the prospective cohort have developed cGvHD so far. First results show that in the majority of the patients the cGVHD markers are different from those forming the aGvHD pattern. The polypeptide patterns for aGvHD and cGvHD will be discussed together with those of other complications, like CMV-reactivation or sepsis.

Taken together our results demonstrate that the proteome analysis of body fluids collected from patients after HSCT maybe extremely useful for diagnosis of complications.