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E. Wagner-Drouet et al.
negative test after the first reactivation (hazard ratio 2.73; P=0.007). Interestingly, a post-hoc analysis con- sidering T-Track® CMV measurements at day 100 after transplantation, a time point highly relevant for outpatient care, showed a positive predictive value of 90.0% in high-risk patients. Our results indicate that standardized cytomegalovirus-specific cellular immunity monitoring may allow improved risk strat- ification and management of recurrent cytomegalovirus reactivation after hematopoietic stem cell trans- plantation. This study was registered at www.clinicaltrials.gov as #NCT02156479.
Introduction
Cytomegalovirus (CMV) infection and disease remain a serious cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT).1–3 Thanks to efficient diagnosis and management of CMV, the inci- dence of death from CMV disease after HSCT has dropped to <10%.4–9 Adequate risk stratification is essential to iden- tify and properly manage patients at risk of CMV reactiva- tion. The main risk factors include donor (D) and recipient (R) CMV serostatus (D-/R+ defining high-risk patients), the use of mismatched or unrelated donors, graft-versus-host disease (GvHD), and intense immunosuppression.1,2 Close monitoring during the first 100 days after transplantation, in accordance with current guidelines, has greatly reduced the incidence of CMV-related complications. However, recur- rent and late-occurring CMV reactivation remain major life- threatening issues, and effective strategies for the preven- tion of late CMV disease prevail as an unmet medical need. This is particularly critical in outpatient care more than 100 days after HSCT when patients are less frequently moni- tored. Studies clearly identified a delay in global and CMV- specific immune reconstitution as a major risk factor for recurrent and late-onset CMV reactivation.10–13 Several CMV-specific immune monitoring assays have been described. They are based on the quantification of the num- ber and/or functionality of immune cells targeted against CMV, using flow cytometry detection, an enzyme-linked immunosorbent assay (ELISA) or enzyme-linked immunospot (ELISpot).14,15 Multiple studies demonstrated the suitability of these methods for predicting recurrent and/or late CMV reactivation, resulting in the emergence of new risk stratification models based on the monitoring of CMV-specific cell-mediated immunity (CMV-CMI) togeth- er with CMV viral load.8,16–25 The lack of standardized assays does, however, render the comparison of most reported results difficult. Two standardized CMV-specific interferon (IFN)-γ ELISpot assays, based on the in vitro stimulation of peripheral blood mononuclear cells (PBMC) with IE-1 and pp65 peptides (T-SPOT®.CMV) or proteins (T-Track® CMV), have been described. T-Track® CMV is highly sensi- tive due to the use of urea-formulated T-activated® IE-1 and pp65 proteins, resulting in the activation of a broad spec- trum of CMV-specific effector cells (including CD4+, CD8+ and NK cells).14,26–28 One study reported the utility of T- SPOT®.CMV to predict the risk of a first treatment-requir- ing CMV reactivation after HSCT.29 Here we describe – to the best of our knowledge for the first time – the utility of the T-Track® CMV assay to predict recurrent and late-onset CMV reactivation after HSCT.
Methods
Study design and participants
A prospective multicenter study was conducted in 175 interme-
diate- and high-risk (D+/R+, D+/R-, D-/R+) allogeneic HSCT recipients. Recipients of a first-time bone marrow or peripheral blood transplantation from either a matched sibling, matched unrelated or mismatched unrelated donor of any gender and race, aged at least 18 years, and scheduled for preemptive antiviral ther- apy were eligible for participation in this study, which was approved by the relevant ethics committees (DIMDI’s registration number 00008544; University of Regensburg’s approval number 13-122-0282). Additional information is provided in the Online Supplementary Methods.
Viral load measurement
CMV load was measured by quantitative polymerase chain reaction using validated, non-standardized protocols and equip- ment (Online Supplementary Methods). CMV reactivation was defined as a CMV viral load requiring antiviral treatment based on center-specific guidelines and/or physician’s decision. Accordingly, the term “CMV reactivation” will thereafter refer to antivirally- treated CMV reactivation.
Measurement and analysis of CMV-specific cell-mediated immunity
Blood collection, peripheral blood mononuclear cell isolation and T-Track® CMV assays (Lophius Biosciences GmbH, Regensburg, Germany) were performed as described26,27,30 and as detailed in the Online Supplementary Methods. IFN-γ ELISpot test results were interpreted on the basis of square-root-transformed spot-forming cells (sqrt-SFC). Briefly, a test was considered posi- tive if the mean of four replicate sqrt-SFC for 200,000 cells (SRM) resulting from IE-1 and/or pp65 stimulation was ≥sqrt(10) and if the difference of the mean of sqrt-SFC of the stimulated condition to that of the unstimulated condition (SRM[stimulated] - SRM[unstimulated]) was ≥0.742.
Lymphocyte subpopulation count determination
Lymphocyte subpopulations were characterized by flow cytometry from the same peripheral blood mononuclear cells and absolute cell counts were calculated using the peripheral blood absolute lymphocyte count determined at the same visit (Online Supplementary Methods).
Statistical analysis
Calculations were performed with SAS 9.4 software, as detailed in the Online Supplementary Methods. Statistical analyses are pre- sented for high-risk (D-/R+) and all patients. Differences in IE-1- and pp65-specific SFC distributions between groups were tested using the Mann-Whitney U test. Qualitative (positive/negative) test results were compared using a χ2 test. The probability of recurrent CMV reactivation according to qualitative test results was estimated using Kaplan-Meier curves. Hazard ratio (HR) esti- mates were obtained by Cox regression analysis and differences in CMV reactivation probability between groups were tested using a log-rank test. Receiver operating characteristic (ROC) curves were generated and area under the curve (AUC) estimates were obtained by logistic regression. Two-sided P-values <0.05 were considered statistically significant.
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haematologica | 2021; 106(2)