Standardized monitoring of cytomegalovirus-specific immunity can improve risk stratification of recurrent cytomegalovirus reactivation after hematopoietic stem cell transplantation

Abstract

Recurrence of cytomegalovirus reactivation remains a major cause of morbidity and mortality following allogeneic hematopoietic stem cell transplantation. Monitoring cytomegalovirus-specific cellular immunity using a standardized assay might improve the risk stratification of patients. A prospective multicenter study was conducted in 175 intermediate- and high-risk allogeneic hematopoietic stem cell transplant recipients under preemptive antiviral therapy. Cytomegalovirus-specific cellular immunity was measured using a standardized IFN-γ ELISpot assay (T-Track® CMV). Primary aim was to evaluate the suitability of measuring cytomegalovirus-specific immunity after end of treatment for a first cytomegalovirus reactivation to predict recurrent reactivation. 40/101 (39.6%) patients with a first cytomegalovirus reactivation experienced recurrent reactivations, mainly in the high-risk group (cytomegalovirus-seronegative donor/cytomegalovirus-seropositive recipient). The positive predictive value of T-Track® CMV (patients with a negative test after the first reactivation experienced at least one recurrent reactivation) was 84.2% in high-risk patients. Kaplan-Meier analysis revealed a higher probability of recurrent cytomegalovirus reactivation in high-risk patients with a negative test after the first reactivation (hazard ratio 2.73; p=0.007). Interestingly, a post-hoc analysis considering T-Track® CMV measurements at day 100 post-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 stratification and management of recurrent cytomegalovirus reactivation after hematopoietic stem cell transplantation. This study was registered at www.clinicaltrials.gov as #NCT02156479.

Figures

Figure 1.

Cytomegalovirus (CMV) viral load at start of the first, second and third CMV reactivations. Median (range) CMV viral load (VL) at the time of the first, second and third CMV reactivations is shown for all patients and high-risk CMV-negative donor/CMV-positive recipient (D-/R+) pairs. It should be emphasized that VL measurements were not standardized among centers. CMV load was measured by quantitative polymerase chain reaction from whole blood (9 centers) or plasma (1 center) using either a commercial assay (Abbott RealTime CMV; 2 centers) or validated in-house protocols and equipment (8 centers). Accordingly, treatment-requiring viral load thresholds were center-specific and no analysis correlating spot-forming-cell counts to VL was planned in this study.

Figure 2.

Legend on following page. Performance of cytomegalovirus (CMV)-specific cell-mediated immunity measured after the end of a first CMV reactivation to predict freedom from and/or occurrence of recurrent CMV reactivation. (A) Interferon-γ enzyme-linked immunospot (ELISpot) was performed after the end of antiviral therapy for a first CMV reactivation, at up to three time points relative to the end of treatment, namely day 0 (d0), day 7 (d7) and day 14 (d14). The first available measurement was considered for the analysis. (B) Quantitative ELISpot results in response to CMV proteins IE-1 and pp65 were evaluated on the basis of the mean of squareroot- transformed (SRM) spot-forming cells (SFC), as described in the Methods section. Differences in SFC distribution between patients with only one CMV reactivation and those with recurrent CMV reactivation were evaluated using a Mann-Whitney U test. Respective P-values are shown under each graph. For the sake of simplicity, scatter plots are depicted as squared SRM values (SRM^2). The median and interquartile range of the SRM^2 SFC are shown above each graph. Additional information (minimum, maximum, 10th and 90th percentiles) are shown in Online Supplementary Table S3. Due to the log scale representation, values of zero SRM^2 were replaced by 0.01 (y-axis), meaning that baseline values shown at y=0.01 are actually equal to zero. Red triangles and blue dots represent negative and positive tests, respectively, defined according to the rules described in the Methods section. Of note, of the three CMV-negative donor/CMV-positive recipients (D-/R+) with a documented recurrent CMV reactivation and with high pp65-SFC after the first CMV reactivation (251 to 386 SFC/200,000 lymphocytes) one was treated for recurrent CMV although the viral load was below the center-specific threshold (0 or 100 copies/mL) in the 10 days preceding the start of treatment and at all time points thereafter; a second patient had a first treatment initiated for a viral load below the center-specific threshold, after which high pp65-specific SFC dropped dramatically over time before the start of treatment of a CMV reactivation with a viral load above the threshold; the third patient had a lengthy (>3 months) first CMV reactivation with a high sustained viral load (up to 110,000 copies/mL), likely reflecting refractory CMV. Importantly, the duration of antiviral therapy for a first CMV reactivation was comparable in patients without and with recurrent reactivation (median [range] duration of 25 [4-94] and 31 [3-77] days, respectively, in all patients [Mann-Whitney U test, P=0.336]; median [range] duration of 31 [4-94] and 34 [7-77] days, respectively, in D-/R+ ptients [Mann-Whitney U test, P=0.677]). (C) Prediction of CMV reactivation recurrence based on IE-1- and pp65-specific SFC counts measured at the end of treatment of a first CMV reactivation was evaluated by receiver operating characteristic curve analysis. Area under the curve estimates, 95% confidence intervals and respective P-values are indicated within each graph. (D) Cumulative probability of CMV reactivation recurrence based on IE-1- and pp65-specific qualitative test results after a first CMV reactivation, evaluated as described in the Methods section. In the case that both IE-1 and pp65 test results are considered (T-TrackR CMV; right panels), a test is positive when at least one IE-1 and/or pp65 test is positive and a test is negative when both IE-1 and pp65 tests are negative. Kaplan-Meier analyses were performed and the respective hazard ratios and P-values are shown within each graph. / indicate censored observations. The median (range) follow-up time after the T-TrackR CMV measurement was 137 (35-180) days in patients with no documented recurrent CMV reactivation (censored). The median (range) time to recurrent CMV reactivation after the T-TrackR CMV measurement was 24 (2-77) days. Moreover, the last recurrent CMV event in the case of a pp65- (and T-TrackR CMV)-positive test result occurred 56 days after the end of antiviral therapy, compared to 77 days in the case of a pp65- and IE-1-negative test result. In (B-D), statistically significant P-values are in bold. CMI: cellmediated immunity; HSCT: hematopoietic stem cell transplantation; IQR: interquartile range; MWU: Mann-Whitney U test; D-/R+: CMV-negative donor/CMV-positive recipient; AUC: area under curve; 95% CI: 95% confidence interval; HR: hazard ratio.

Figure 3.

Legend on following page. Performance of cytomegalovirus (CMV) cell-mediated immunity measured around day 100 after hematopoietic stem cell transplantation to predict freedom from and occurrence of late recurrent CMV reactivation (post-hoc). (A) Interferon-γ enzyme-linked immunospot (ELISpot) tests performed between day 80 (d80) and day 100 (d100) after hematopoietic stem cell transplantation in patients with an earlier CMV reactivation were used for the analysis. In the case that several measurement were available, the one closest to day 100 was considered. (B) Quantitative ELISpot results in response to CMV IE-1 and pp65 proteins were evaluated as detailed in the legend to Figure 2. Differences in spot-forming cell (SFC) distribution between patients with a CMV reactivation before day 100 only and those with a late (after day 100) recurrent CMV reactivation were evaluated using a Mann-Whitney U test. The median and interquartile range of the squared mean of square-root-transformed (SRM^2) SFC are shown above each graph. Additional information (minimum, maximum, 10th and 90th percentiles) are shown in Online Supplementary Table S4. Due to the log scale representation, values of zero SRM^2 were replaced by 0.01 (y-axis), meaning that baseline values shown at y=0.01 are actually equal to zero. Red triangles and blue dots represent negative and positive tests, respectively, defined according to the rules described in the Online Supplementary Methods. (C) Prediction of late recurrent CMV reactivation based on IE-1- and pp65-specific SFC counts measured around day 100 was evaluated by receiver operating characteristic curve analysis, as detailed in the legend to Figure 2. (D) Cumulative incidence of recurrent CMV reactivation based on IE- 1- and pp65-specific qualitative test results around day 100, was evaluated as detailed in the legend to Figure 2. Median (range) follow up time after T-TrackR CMV measurement was 132 (59-145) days in (censored) patients with no documented late recurrent CMV reactivation. The median (range) time to late recurrent CMV reactivation after T-TrackR CMV measurement was 119 (105-165) days. Moreover, the last recurrent CMV event in the case of a pp65- (and T-TrackR CMV)-positive test result occurred 31 days after the day-100 measurement, compared to 77 days in the case of a pp65- and IE-1-negative test result. CMI: cell-mediated immunity; HSCT: hematopoietic stem cell transplantation; IQR: interquartile range; MWU: Mann-Whitney U test; D-/R+: CMV-negative donor/CMV-positive recipient; AUC: area under curve; 95% CI: 95% confidence interval; HR: hazard ratio.