IFN-γ induced by PHA stimulation as new marker for GVHD prediction in patients undergoing Allogeneic Hematopoietic Stem Cell Transplantation

Abstract

Background: IFN-γ is crucial in the pathogenesis of GVHD and higher levels are reported to be elevated in patients with active chronic GVHD. Immune-monitoring of INF-levels after alloHSCT could help in the management of GVHD. A recent ELISA based test (QuantiFERON®-CMV) could measure specific (anti-CMV) and aspecific production of IFN-γ in whole blood. Preliminary data suggests that aspecific production of IFN-γ should be associated with GVHD. The aim of this study is to confirm the reliability of QuantiFERON®-CMV for association and prediction of GVHD. Methods: the study was performed in 2 phases. In the fisrt phase of the study, 92 whole blood specimen were collected and analyzed from 29 patients undergoing alloHSCT in order to confirm the preliminary data. QuantiFERON® CMV is an in vitro diagnostic test that use an antigenic human cytomegalovirus proteins (CMV) peptide cocktail to stimulate cells from whole blood. Detection of interferon-γ (IFN-γ) by ELISA is used to identify responses to these peptide antigens. The IFN-γ response in the CMV Ag tube is considered positive if > 0.2 UI/mL as defined by the manufacturer. The Mitogen-stimulated (PHA) plasma sample is used as an IFN-γ positive control for each specimen tested. In order to assess the association between IFN-γ response due to PHA stimulation and GVHD, the positivity of the test was determined according to 2 different cut-off: #1) 0,5 IU/mL as defined by manufacturer, #2) 9 IU/mL as experimentally defined by the median of the observations in our data set. GVHD extension was defined by Seattle criteria and/or the number of involved sites, Chi-square test was used to assess the statistical correlation between IFN-γ production and clinical outcomes. In the second phase 10 patients were observed prospectively with collection of blood samples every 2-3 weeks since engraftment until 4-6 months after SCT in order to study the PHA stimulated IFN-γ production in relationship with the onset of chronic GVHD. Results: among 92 samples 70 were positive for the PHA stimulated IFN-γ production according to the cut-off #1; 61% (43/70) were associated with GVHD whereas 27% (6/22) with lower PHA stimulated IFN-γ production were associated with GVHD: this difference was proved to be significant (p=0.005). Among 92 samples 46 were positive for the PHA stimulated IFN-γ production according to the cut-off #2; 71% (33/46) were associated with GVHD whereas 34% (16/46) with lower PHA stimulated IFN-γ production were associated with GVHD: this difference was proved to be significant (p=0.000). Among the 10 patients observed prospectively during the first 6 months after alloHSCT 7 became positive for the PHA stimulated IFN-γ production: 6/7 developed GVHD in a median time of 100 days according to the cutoff #1 and after a median time of 33 days according the cutoff#2. Four patients received steroid treatment for extensive chronic GVHD and their PHA stimulated IFN-γ production dropped after treatment (figure 1). Conclusions: The PHA stimulated IFN-γ production is strictly associated to GVHD, seems to predict its onset and could help in the modulation of immunesuppressive treatment. However, larger prospective studies are needed