Page 54 - 2021_02-Haematologica-web

E. Wagner-Drouet et al.
Figure 2 (previous page). Performance of cytomegalovirus (CMV)-specific cell-mediated immunity measured after the end of a first CMV reactivation to predict free- dom 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 measure- ment 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 square- root-transformed (SRM) spot-forming cells (SFC), as described in the Methods section. Differences in SFC distribution between patients with only one CMV reactiva- tion 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 sim- plicity, 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 recur- rent 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 dramati- cally 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 reac- tivation with a high sustained viral load (up to 110,000 copies/mL), likely reflecting refractory CMV.55 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-Track® 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-Track® 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-Track® CMV measurement was 24 (2-77) days. Moreover, the last recurrent CMV event in the case of a pp65- (and T-Track® 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: 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.
Table 3. Diagnostic accuracy in identifying patients with and without recurrent cytomegalovirus (CMV) reactivation based on CMV-specific negative and positive enzyme-linked immunospot test results after the first CMV reactivation.
Population
All patients
D-/R+ patients
Marker
IE-1 pp65 IE-1, pp65a IE-1
pp65 IE-1, pp65a
Sensitivity
73.3% (22/30) [95% CI: 54.1-87.7%]
58.1% (18/31) [95% CI: 39.1-75.5%]
53.3% (16/30) [95% CI: 34.3-71.7%]
75.9% (22/29) [95% CI: 56.5-89.7%] 58.6% (17/29) [95% CI: 38.9-76.5%] 55.2% (16/29) [95% CI: 35.7-73.6%]
Specificity
56.8% (25/44) [95% CI: 41.0-71.7%]
88.4% (38/43) [95% CI: 74.9-96.1%]
93.2% (41/44) [95% CI: 81.3-98.6%]
17.7% (3/17) [95% CI: 3.8-43.4%] 77.8% (14/18) [95% CI: 52.4-93.6%] 83.3% (15/18) [95% CI: 58.6-96.4%]
Chi-square
P=0.010
P<0.001
P<0.001
P=0.606 P=0.015 P=0.009
PPVb NPVb
-- -- --
61.1% (22/36) 80.9% (17/21) 84.2% (16/19)
30.0% (3/10) 53.8% (14/26) 53.6% (15/28)
aT-Track® CMV assay: the test is positive when at least one of the IE-1- and/or pp65-specific response is positive, and the test is negative when both IE-1- and pp65-specific responses are negative; bPositive and negative predictive values were not calculated in the “all patients” population because of the imbalance toward CMV-negative donor/CMV-positive recipient patients in that group. 95% CI: 95% confidence interval; CMV: cytomegalovirus; D/R: donor/recipient CMV serostatus; NPV: negative predictive value; PPV: positive pre- dictive value.
2D). Interestingly, the performance of the pp65 test was improved by the combination with IE-1 (T-Track® CMV test), in all patients (HR 5.68; log-rank test, P<0.001) and in D-/R+ patients (HR 2.73; log-rank test, P=0.007) (Figure 2D).
To better understand the usability of the assay in terms of clinical cutoff, we evaluated the PPV (patients with SFC ≤ threshold after the first CMV reactivation had a recur- rent CMV reactivation) and NPV (patients with SFC > threshold after the first CMV reactivation did not have a recurrent CMV reactivation) of pp65-specific response in D-/R+ patients at low, intermediate and high SFC counts (Table 4). A NPV of 100% (3/3) was observed for a thresh- old of 386 SFC/200,000 lymphocytes in association with a PPV of 65.9% (29/44). A NPV of 75.0% (9/12) and PPV of 74.3% (26/35) were found for a SFC of 40 SFC/200,000 lymphocytes. Higher PPV (87.5% [21/24] to 100% [8/8]) and lower NPV (65.2% [15/23] to 46.2% [18/39]) were observed at lower (9 and less) SFC counts (Table 4), in line with the results described above (Table 3).
Benefit of monitoring CMV-specific cell-mediated immunity over absolute T-cell counts
We next compared the performance of CMV-CMI to that of absolute lymphocyte (T and NK cells) counts meas- ured after end of treatment of a first CMV reactivation to predict subsequent CMV reactivation episodes. Multicolor flow cytometry was performed using remain- ing peripheral blood mononuclear cells. NK and T (total, naïve and memory) cell levels were expressed as absolute cell counts. The first visit with existing absolute cell counts following the end of antiviral therapy (of day 0, 7 and 14) was considered for the analysis. Absolute cell counts were significantly higher in patients with no recur- rent CMV reactivation in all cases, except for total and memory CD4+ T cells in D-/R+ patients (Online Supplementary Figure S8A). In ROC analyses, total lympho- cytes as well as total and naïve CD8+ cell populations showed a good predictive value for recurrent CMV reacti- vation, with AUC estimates between 0.813 and 0.833 in D-/R+ patients (Online Supplementary Figure S8B), thus in a
368
haematologica | 2021; 106(2)