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Risk stratification of CMV recurrence after HSCT
Table 4. Positive and negative predictive values of low, intermediate and high spot-forming-cell counts of a pp65-specific enzyme-linked immunospot assay after the end of treatment for a first cytomegalovirus (CMV) reactivation to predict the occurrence of future recurrent CMV reactivation in high-risk hematopoietic stem cell transplant patients.
Patient population
Marker
Thresholda
386
40
9
0
PPV
65.9% (29/44)
74.3% (26/35)
87.5% (21/24)
100% (8/8)
NPV
100% (3/3)
75.0% (9/12)
65.2% (15/23)
46.2% (18/39)
% patients above threshold
6.4% (3/47)
25.5% (12/47)
48.9% (23/47)
83.0% (39/47)
D-/R+ pp65
aSFC (SRM^2)/200,000 lymphocytes (stimulated minus unstimulated condition) rounded to closest spot count; thresholds were derived from Receiver operating characteristic curve data; the threshold of 9 SFC (SRM^2)/200,000 lymphocytes showed the highest sensitivity+specificity value. SFC: spot-forming cells; SRM^2: squared mean of square-root- transformed; CMV,: cytomegalovirus; D/R: donor/recipient CMV serostatus; NPV: negative predictive value; PPV: positive predictive value.
comparable range to that of the AUC derived from pp65- specific ELISpot test results (Figure 2C and Online Supplementary Figure S5C). NK cells as well as total, naïve and memory CD4+ T-cell subpopulations showed a lower predictive value, especially in D-/R+ patients, with AUC estimates between 0.615 and 0.729 (Online Supplementary Figure S8B). A ROC analysis of paired pp65-specific ELISpot results and absolute lymphocyte and T-cell counts obtained at the same visit after the end of therapy showed no statistically significant difference in predictability, although pp65-specific ELISpot results normalized to absolute lymphocyte counts tended to perform better than absolute cell counts (Online Supplementary Figure S8C).
CMV-specific cell-mediated immunity at day 100 post-transplantation can predict late recurrent CMV reactivation (post-hoc analysis)
Accurate prediction of future recurrent CMV reactiva- tion is particularly critical when patients are released from close monitoring in an outpatient setting around 3 months after HSCT.4–6 We conducted two post-hoc analyses to determine whether CMV-CMI monitoring at the fixed time of day 100 could identify patients at risk of future (i.e. late) CMV reactivation. The first analysis considered all patients, regardless of a possible existence of CMV reactivation prior to day 100, thus assessing occurrence of late CMV reactivation generally (Online Supplementary Figure S9). The second analysis focused on patients who experienced CMV reactivation prior to day 100, thus investigating the usefulness of the IFN-γ ELISpot meas- ured around day 100 to predict late recurrent CMV reacti- vation (Figure 3). T-Track® CMV test results acquired between day 80 and day 100 after transplantation were considered in patients with no ongoing CMV reactivation (Online Supplementary Methods).
Interestingly, patients (including those in the high-risk group) who did not experience CMV reactivation up to day 80-100 did not experience CMV reactivation there- after (Online Supplementary Figure S9B, orange-labeled dots). A majority of these patients presented low IE-1 and pp65-specific test results throughout the study (Online Supplementary Figure S9B and data not shown). Such sus- tained low responsiveness in patients with no CMV reac- tivation was previously reported.33 Its cause remains to be investigated. Consequently, performance of the ELISpot test at day 100 in this global analysis of late CMV reacti- vation was low (Online Supplementary Figure S9B-D; Online Supplementary Table S5). On the other hand, the analysis of ELISpot test results at day 100 in patients with an earlier CMV reactivation (Figure 3A) revealed a significant differ- ence in SFC counts between patients without and with
late recurrent CMV reactivation (Mann-Whitney U test, P- value between 0.013 and <0.001), with the exception of IE-1-specific response in D-/R+ patients (Mann-Whitney U test, P=0.277) (Figure 3B). All patients with late recur- rent CMV reactivation belonged to the high-risk group. In ROC analyses, AUC estimates for pp65 test results were 0.811 (P=0.003) in the D-/R+ population and 0.911 (P<0.001) in the total population (Figure 3C). pp65-specific response showed the best diagnostic accuracy, with a sen- sitivity and specificity of 75.0% and 90.9%, and a PPV and NPV of 90.0% and 76.9% in the D-/R+ population, respectively (Table 5). The probability of late recurrent CMV reactivation was significantly higher in D-/R+ patients with a pp65-negative test at day 100 (HR 6.34; log-rank test, P=0.002). In this setting, the response to IE- 1 did not improve the performance of pp65 (Figure 3D). Remarkably, the probability of recurrent CMV in the case of pp65- and T-Track® CMV-positive tests was lower when measured at day 100 than when measured directly after a first CMV reactivation (compare blue curves in Figures 2D and 3D).
Discussion
This study demonstrates for the first time the suitability of a standardized CMV-specific IFN-γ ELISpot assay to predict recurrence of CMV reactivation after HSCT, with a particular focus on clinically relevant high-risk D-/R+ patients.
Overall, the response to pp65 antigen in the ELISpot assay was higher than that of IE-1, as previously report- ed.26,27,30 Although the IE-1-specific response had low or no predictive value alone, it improved the assay performance in combination with pp65-specific tests after the first CMV reactivation.
Diagnostic accuracy and time-to-event analyses indicat- ed that negative IFN-γ ELISpot test results could predict recurrence of CMV reactivation when measured after the end of anti-CMV therapy or around day 100 after HSCT. Positivity of the T-Track® CMV test is based on several rules, the main one being that CMV antigen-stimulated conditions must yield ≥10 SFC (or mean of sqrt SFC ≥3.16) (Online Supplementary Methods). This raises the possibility that the technical cutoff of the assay might be a relevant clinical cutoff for the prediction of recurrent CMV reacti- vation (PPV >80%). Whether the positivity cutoff of T- Track® CMV is indeed a valid clinical cutoff to predict recurrent CMV reactivation in high-risk D-/R+ patients could be addressed in a randomized interventional study. In fact, such an approach is currently being appraised in high-risk solid-organ transplant recipients in a study aim-
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