IFN-γ induced by PHA stimulation as new marker for GvHD prediction in patients undergoing allogeneic hematopoietic stem cell transplantation (alloHSCT)

Introduction GVHD is associated with a high morbidity and mortality in alloSCT patients. An early diagnosis of GVHD could reduce this adverse impact on the outcome of alloSCT. The effect of Th1 cytokine IFN-γ is crucial in the pathogenesis of GVHD and, as expected, higher protein levels are reported in the serum of patients with active chronic GVHD. Aims and Methods Our hypotesis is that the monitoring of IFN-γ basal levels as well as IFN-γ induced by mitogen stimulation in the blood samples of patients after alloSCT could help the management and the prediction of GVHD. A recent ELISA based test (QuantiFERON®-CMV) could measure specific (anti-CMV) and aspecific production of IFN-γ in whole blood. The aim of this study is to assess the reliability of the positive control of the QuantiFERON®-CMV kit as new marker for GVHD early diagnosis during immune system reconstitution after bone marrow transplantation. Methods: QuantiFERON®‑CMV is an in vitro diagnostic test that use an antigenic human cytomegalovirus proteins (CMV) peptide cocktail to stimulate cells from whole blood and is used after alloSCT to monitor the occurrence of CMV infection. The mitogen-stimulated (PHA) plasma sample is used as a positive control for each specimen tested. Detection of interferon-γ (IFN-γ) by ELISA is used to identify responses. In order to assess the association between PHA stimulation IFN-γ production and GVHD, we decided to consider 3 different positivity of the test: 1) 0,5 IU/mL as defined by manufacturer, 2) 9 IU/mL as experimentally defined by the median of the observations in our preliminary data set. 3) 5 IU/mL as defined by the median of the values of the prospective study. GVHD extension was defined by Seattle criteria and/or the number of involved sites. A prospective study has been designed in order to confirm these data. Thirty-six patients were prospectively monitored after transplant with QuantiFERON®‑CMV according the following timepoints: every 2-3 weeks until +180d since transplantation, every month until +365d. Lymphocyte subpopulations +28, +56, +84, every month thereafter. Events (acute GVHD, chronic GVHD, relapse, death are registered in the database and matched with the production of PHA induced INF-y; when GVHD was suspected a biopsy of the target organ was recommended. A two step cox regression model for time dependent variables was implemented in order to define risk factors for cGvHD. Results PHA-induced INF-Y increase in the first three months after alloHSCT was independently associated with cGvHD at the multivariate analysis (p= 0.021, 0.015, 0.027 respectively) together with recipient sex (female), age, stem cell source, GvHD prophylaxis and infections. The sensitivity of the test was 97% at the cutoff 1 (0.5 IU/mL). The reduction of PHA-induced INF-Y was detected in all patients treated for a cGvHD but one who did not respond to immune-suppression. Conclusions PHA-induced INF-Y is a reliable biomarker of cGvHD and could help the physician in the management of cGvHD treatment. Further studies are needed in order to better define subsets of patients who could benefit from this test.