Evaluation of Virus-Specific T Cell Responses in Patients Affected with Myeloproliferative Neoplasms Treated with Ruxolitinib
Authors: Rumi Elisa , Cavalloni Chiara , Comolli Giuditta , Ferretti Virginia Valeria , Cassaniti Irene , Pietra Daniela , Trotti Chiara , Ciboddo Michele , Degli Antoni Melania , Furione Milena , Baldanti Fausto , Arcaini Luca , Cazzola Mario 
Published: November 2019

Background The classic Ph-negative myeloproliferative neoplasms (MPN) are a group of clonal haematopoietic disorders, including polycythemia vera (PV), essential thrombocythemia (ET) and myelofibrosis (MF) (either primary or secondary), that share the deregulation of JAK-STAT signalling. Over the past few years, there have been significant changes in the therapeutic landscape of MPN, thanks to the approval of the JAK-inhibitor ruxolitinib. Despite its efficacy and beyond its well described haematological toxicity, the drug may also cause an increased risk of reactivation of silent infections (e.g., tuberculosis, hepatitis B virus and varicella zoster virus). Less is known regarding other opportunistic viral pathogens, such as human cytomegalovirus (HCMV) and Epstein-Barr virus (EBV). The aim of this study was to evaluate the viral load and T cell responses to HCMV and EBV in 25 MPN patients (6 PV, 5 ET, 14 MF) treated with ruxolitinib.
Methods Peripheral blood was collected monthly for viral genome quantification using real-time PCR (EBV-DNA and HCMV-DNA) and determination of T cell subsets by flow cytometry (absolute number of CD3+, CD3+CD4+, and CD3+CD8+). The T cell responses specific to HCMV and EBV were evaluated monthly using IFN-γ ELISPOT assay. Results were normalized to CD4+ and CD8+ T cell count. Correlations between T cell responses were evaluated by regression models for panel data (with random or fixed effects based on result of Hausman test). P-values <0.05 were considered significant.
Results Of 25 patients treated with Ruxolitinib (median time of treatment: 5 years, range: 0.2-10.5 years), 15 (60%) had CD4+ T cells and 7 (28%) had CD8+ T cells below normal ranges. The reduction was observed in all MPN subtypes: CD4+ T cells were reduced in 50% of PV, 80% of ET, 57% of MF; CD8+ T cells were reduced in 33% of PV, 60% of ET and 14% of MF. The reduction was not different based on disease duration (<10 years vs >= 10 years since diagnosis) while it correlated with duration of ruxolitinib treatment: patients receiving ruxolitinib for more than 5 years had more frequently a reduction of CD4+ T cells and/or CD8+ T cells compared with patients treated for less than 5 years (93% vs 45% P 0.021). Moreover, the median number of CD4+ cells and CD8+ cells were lower in patients treated >= 5 years vs <5 years (366 vs 558 cells/μl P = 0.043 and 206 vs 365 cells/μl P = 0.002). During the study, reactivation of EBV was observed in 76% of patients while only 8% experienced reactivations of HCMV ; only one patient (4%) experienced Varicella Zoster Virus clinical reactivation. Both the EBV-specific and CMV-specific CD4+ and CD8+ T cell responses showed an inverse correlation with detectable EBV DNA and HCMV DNA although it was not statistically significant (all p-values>0.2). When normalizing results to CD4 and CD8 T cell counts we observed again an inverse correlation without statistical significance (all p-values>0.1). Both the EBV-specific and CMV-specific CD4+ and CD8+ T cell responses had an inverse correlation with the dosage of ruxolitinib (<= 20 mg/die vs > 20 mg/die) although not statistically significant (all p-values>0.06).
Conclusions Our study suggests that a reduction of CD4+ T cells and CD8+ T cells is frequently observed in MPN patients treated with ruxolitinib and is associated with treatment duration (>= 5 years vs <5 years). Reactivation of EBV occurs frequently while reactivation of CMV is less frequent. As the virus-specific T cell responses seem to have a trend to an inverse correlation with detectable EBV DNA and HCMV DNA we hypothesize that an impaired response makes the patient unable to clear the virus. Moreover, the virus-specific T cell responses seem to have a trend to an inverse correlation with ruxolitinib dosage, thus pointing to a potential role of the drug in favouring viral reactivation. To corroborate this hypothesis, virus-specific T cell responses need to be analysed in a larger number of patients with MPN under ruxolitinib treatment.

Disclosures
Rumi: novartis: Honoraria, Research Funding. Arcaini:Gilead Sciences: Research Funding; Celgene: Speakers Bureau; Bayer, Celgene, Gilead Sciences, Roche, Sandoz, Janssen-Cilag, VERASTEM: Consultancy; Celgene, Roche, Janssen-Cilag, Gilead: Other: Travel expenses.