INTRODUCTION

Cytomegalovirus (CMV) research in the last decade has been almost solely focused on the utility and importance of quantitative polymerase chain reaction (qPCR) to detect and treat CMV DNAemia after an allogeneic hematopoietic cell transplant (HCT) or solid organ transplant (SOT). More recently, the role of CMV-specific cellular mediated immunity (CMV CMI) has emerged as an important component affecting CMV DNAemia posttransplant, with active research ongoing in both HCT and SOT recipients [1]. In contrast, the utility of CMV serology has remained unchanged through decades, predominately associated with donor selection and posttransplant prophylactic and monitoring strategies [2–4]. The last two years have seen a renewed interest in the importance of CMV immunoglobulin G ( IgG) titers in allogeneic HCT recipients, suggesting that a dichotomous interpretation of CMV serology as positive or negative may not necessarily be an accurate and sufficient interpretation. In contrast, quantification of CMV IgG titers may allow for correct interpretation of CMV status of allogeneic HCT recipients and predict CMV DNAemia posttransplant [5▪▪,6,7▪▪,8,9▪,10▪]. Herein, we report on the most recent data on the importance of CMV IgG titers in the allogeneic HCT setting and future perspectives. 

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CYTOMEGALOVIRUS IMMUNOGLOBULIN G TITERS AND CYTOMEGALOVIRUS RECIPIENT STATUS

In most transplant centers, CMV serology results are usually presented as a dichotomous variable (positive vs. negative), not allowing for further interpretations. Furthermore, CMV “indeterminate” serology results have been reported in the literature, without clarifying what this “indeterminate” status may indicate. It has been suggested that multiple blood product transfusions in hematology patients may lead to false positive CMV serology results, due to the presence of CMV IgG in the transfused products [11,12]. Henceforth, the British Society of Hematology has proposed baseline CMV serology testing for all hematology patients who are likely to proceed to an allogeneic HCT, in order to document their CMV serology status prior to blood product transfusions [13]. The question on “indeterminate” CMV serology results and potentially falsely-labeled CMV R+ following blood product transfusions has remained open for decades. Recent data from our group suggest that almost 30% of allogeneic HCT recipients may be mislabeled as CMV R+, while in fact they are CMV R− [5▪▪]. In a single-center cohort study over the period of 5 years including 291 consecutive adult allogeneic HCT recipients, 246 (84.5%) patients were identified as CMV R+ on their pretransplant routine CMV serology testing. Amongst them, 180 (73%) patients had a positive or unknown CMV serology at the time their underlying hematology malignancy was diagnosed and were therefore excluded from all further analyses. The remaining 66 (27%) patients had a negative CMV serology at the diagnosis of their hematology malignancy. After excluding six patients (2, 2, and 2 due to CMV DNAemia at the time of their pretransplant routine CMV serology testing, CMV IgG titer >500 IU/ml, and nonreclassification, respectively), there were 60/246 (27%) patients remaining, with negative CMV serology at the time their underlying hematology malignancy was diagnosed, and indeterminate (N: 10; CMV IgG titer ≥0.6 to ≤3 IU/ml) or low-positive IgG titer (N: 50; CMV IgG titer >3 to <50 IU/ml) CMV serology, and negative CMV DNAemia at the time of their pretransplant routine CMV serology testing. Those patients were reclassified from CMV R+ to CMV R− (CMV R− reclassification group). When compared to the remaining 44 patients with negative CMV serology (CMV IgG titer <0.6 IU/ml; CMV R− control group) both at the time of diagnosis of their underlying hematologic malignancy and their pretransplant routine CMV serology testing (excluding one patient with CMV DNAemia detected at the pretransplant routine CMV serology testing), 1/60 (1.67%) and 3/44 (6.8%; P = 0.30) in the CMV R− reclassification and CMV R− control group developed CMV DNAemia during the first 6 months posttransplant. The results of this study are limited as they are based on the interpretation of a single serology test (Elecsys CMV IgG, Roche Diagnostics, Rotkreuz, Switzerland) with a cutoff for negative, indeterminate, and positive results at: <0.6, ≥0.6 to ≤3, and >3 IU/ml, respectively (with a range of quantification between 0 and 500 IU/ml). The definition of “low-positive” CMV IgG titers between 3 and 50 IU/ml was arbitrary and driven by the clinical experience of the investigators, rather than being based on definitive data. In addition, data on blood transfusions between diagnosis of hematology malignancy and HCT were not always available. Finally, this study does not answer the question on the significance of low-positive CMV IgG titers when baseline CMV serology results at the time of hematologic malignancy diagnosis are not available. Nevertheless, those data suggest that almost one third of allogeneic HCT recipients labeled as CMV R+ could in fact be CMV R−, with significant clinical and logistical implications, in terms of donor selection and exposure to letermovir prophylaxis. In our study, reclassification of those 60 patients from CMV R+ to CMV R− resulted in saving more than $2 million associated with primary anti-CMV prophylaxis. While more data are required to further validate those observations, a simple approach including baseline CMV serology testing at the time of hematologic malignancy and a more careful read of CMV IgG titers at pretransplant screening may have significant implications for our patients and institutions.

PRE-TRANSPLANT CYTOMEGALOVIRUS IMMUNOGLOBULIN G TITER AS A PREDICTOR OF POST-TRANSPLANT CYTOMEGALOVIRUS REPLICATION

Recent results suggest that allogeneic HCT CMV R+ may be at different risk for CMV DNAemia posttransplant, based on the degree of positivity of their CMV IgG titer [6,7▪▪,8,9▪,10▪]. Although the underlying pathophysiology remains to be defined and better clarified in the future, it has been hypothesized that higher CMV IgG titers in the recipient may indicate a higher burden of subclinical CMV activity of the viral latent reservoir, rather than a correlate of protection. Recent data from five centers from the North and South America, Japan, and Europe underscoring the significance of pretransplant CMV IgG titer in posttransplant CMV replication in this population have been published or presented in the last 4 years (Table 1) [6,7▪▪,8,9▪,10▪].

Table 1 - Summary of studies reporting on the association between pretransplant CMV humoral immunity and CMV reactivation after allogeneic hematopoietic cell transplant Study Study type Study period Patient population CMV serology assay CMV replication test CMV serology cutoffs Association CMV IgG - CMV DNAemia Association CMV IgG − outcomes Arcuri L et al. Brazil [6] Cohort, single-center 2015–2020 98 Allo-HCTr DiaSorin LIAISON Kit CMV pp65 Antigen CMV qPCR 100 IU/ml 100-day CMV reactivation incidence higher in patients with CMV IgG >100 U/ml (63% vs. 31%; P = 0.004) NA Kawamura S et al., Japan [7▪▪] Cohort, single-center 2007–2017 309 Allo-HCTr EIA (SRL, Tokyo, Japan) CMV pp65 Antigen <2 IU/ml (negative)
2–5.8 IU/ml (low)
≥5.8 IU/mL (high) “High titer” group more likely to reactivate CMV by D180 (81%) compared to “low titer” (37%) and “negative titer” (16%) groups (P < 0.01) No difference in relapse or NRM. Trend for higher mortality and aGvHD by D100 in “high titer” group. Eberhardt K et al., Cologne [8] Cohort, single-center 2008–2019 440 Allo-HCTr Abbott Whole blood CMV qPCR 0–5 AU/ml (A)
6–249 AU/ml (B)
≥ 250 AU/mL (C) Higher pretransplant CMV-specific humoral immunity titers associated with higher incidence of CMV DNAemia (any and ≥150 IU/ml) at D100 3-year mortality was higher in group A (74%) compared to group B (47%; P = 0.002) Royston L et al., Geneva [9▪] Cohort, single-center 2010–2023 422 Allo-HCTr Abbott (2010–2013) Elecsys (2013–2023) Plasma CMV qPCR 3–50 IU/ml >50 IU/ml CMV IgG titers ≥50 IU/mL associated with higher rates of CMV DNAemia (P < 0.001) by D180 NA Zamora D et al., Seattle [10▪] Cohort, single-center 2007–2017 478 Allo-HCTr Phage Display immunoprecipitation and sequencing (VirScan) Plasma CMV qPCR CMV-specific antibody metrics categorized into quartiles Cumulative incidence of any CMV reactivation at D100 lower in patients in 1st quartile (P < 0.001) NA

aGvHD, acute graft-versus-host disease; allo-HCTr, allogeneic hematopoeitic cell transplant recipients; AU, arbitrary units; CMV, cytomegalovirus; cs, clinically significant; D, day post-HCT; EIA, enzyme immunoassay; NA, not applicable; NRM, non-relapse mortality; qPCR, quantitative polymerase chain reaction.The antigen–antibody strength was assessed by measuring both CMV epitope hits (mean epitope binding signal, EBS; for all 478 patients) and CMV neutralizing antibodies for 174 patients.

In a first retrospective single-center cohort from Brazil published in 2020, 68 HCT adult recipients with unrelated donors and 30 with haplo-identical donors were included [6]. With a median pretransplant anti-CMV IgG titer of 109 U/ml and a median follow-up of 2.2 years, the authors found that the incidence of CMV reactivation (defined as any positive CMV pp65 antigenemia or any quantitative real-time PCR result >100 copies/ml) was higher in patients with anti-CMV IgG level >100 U/mL (100-day cumulative incidence of 63% vs. 31%; P = 0.004). This was also confirmed in a multivariate analysis, with anti-CMV IgG levels >100 U/ml being associated with the first CMV reactivation event (HR, 2.38; P = 0.005). This finding was unexpected as the investigators’ initial hypothesis was that the risk of reactivation would be lower in patients with higher anti-CMV IgG titers. In this first exploratory study, other clinical outcomes were not studied.

In a retrospective, single-center cohort study from Japan, 309 consecutive adult allogeneic HCT recipients transplanted between 2007 and 2017 were included [7▪▪]. Patients with primary engraftment failure and those who received primary CMV prophylaxis with letermovir were excluded. Pretransplant CMV serology was measured with an enzyme immunoassay (EIA) with a positivity cutoff of 2.0 (SRL, Tokyo, Japan). Using ROC analysis results of the CMV IgG titer, patients were divided in “high”, “low”, and “negative” titer for a CMV IgG titer ≥5.8, 2–5.8, and <2, respectively. CMV replication was measured with weekly CMV antigenemia, using the pp65C10/11 method, with different thresholds for preemptive treatment initiation based on the type of HCT and conditioning regimen administered. The incidence of CMV infection during the first 6 months posttransplant was assessed, followed by disease relapse, overall and nonrelapse mortality (NRM), and acute grade ≥2 GvHD incidence. Notably, there were 6% of patient who received a cord-blood HCT and thymoglobulin and alemtuzumab were administered in 12% and 8% patients, respectively. Patients in the “high titer” group were more likely to reactivate CMV by 180 days posttransplant (81%) compared to the “low titer” (37%) and “negative titer” (16%) groups (P < 0.01). Similarly, risk factor analysis identified a pretransplant high CMV titer as a significant predictor of CMV reactivation (hazard ratio, HR 9.31, 95% confidence interval, CI 4.16, 20.84, P < 0.01). A CMV IgG titer ≥5.8% was considered as an important cutoff above which 80% of patients would reactivate CMV posttransplant. Notably, 15 cases of CMV disease were identified in the cohort, all in the “high titer” group. Although no difference across the three different CMV IgG titer groups in disease relapse or 2-year NRM was observed, there was a trend for higher 2-year mortality and acute grade ≥2 GvHD incidence by day 100 posttransplant in the “high titer” group. This study is the first to report on the clinical significance of pretransplant CMV IgG titer on posttransplant clinical outcomes, although limited by the relatively small number of patients, use of CMV antigenemia for the detection of CMV replication, and a CMV serology assay not widely used in the rest of the world.

Another single-center cohort study was conducted between 2008 and 2019, including 440 consecutive adult allogeneic HCT recipients [8]. CMV IgG were measured using the Abbott Architect CMV IgG chemiluminescent microparticle immunoassay (CMIA; Abbott, Abbott Park, IL, USA), with a positivity cutoff of ≥6 arbitrary units (AI)/ml and a lower (L) and upper level of quantification (ULOQ) of 0 and 250 AU/ml, respectively. Patients were divided in three groups based on their pretransplant CMV IgG titer: A, B, and C, for a CMV IgG titer of ≥250 AU/ml, 6–249 AU/ml, and 0–5 AU/ml, respectively. An intensive CMV DNAemia monitoring on whole blood was routinely performed in that center, twice weekly during the first 100 days and once weekly thereafter for the first-year posttransplant. Clinically significant (cs) CMV infection was defined as any CMV DNAemia leading to initiation of preemptive CMV treatment, routinely started with CMV DNAemia titers >2000 IU/ml once or >1000 IU/ml twice. Overall, patients in group A were more likely to develop csCMV infection posttransplant (67%) compared to patients in group B (51%, P = 0.036). Similarly, 3-year mortality was higher in group A (74%) compared to group B (47%; P = 0.002). There was no difference in 1-year mortality and the peak titer and days of CMV DNAemia between groups A and B.

Our group has previously reported on the importance of pretransplant CMV IgG titers in allogeneic HCT R+ for posttransplant CMV DNAemia and csCMV infection [9▪]. In a single-center 13-year cohort study, we reviewed the impact of pretransplant CMV IgG titers on CMV replication during the first 180 days posttransplant in 422 allogeneic HCT CMV R+ (294 D+/R+ and 128 D−/R+). The median CMV IgG titer was 65.4 IU/ml (IQR 3, 200) in the whole cohort, and 58.6 IU/ml (IQR 2.4, 200) and 79.4 IU/ml (IQR 4.2, 347) in the pre- and postletermovir era. In the overall cohort, pretransplant recipient CMV IgG titers ≥50 IU/ml were associated with higher rates of CMV DNAemia (any, P < 0.001; >150 IU/ml, P < 0.001; >500 IU/ml, P < 0.001) in the first 6 months posttransplant. Similar results were observed in the postletermovir cohort for any (P < 0.001), >150 IU/ml (P = 0.001), and >500 IU/ml (P = 0.009) CMV DNAemia. Those results have only been presented in an abstract form so far. The clinical significance of those findings may be limited by the single-center retrospective nature of this study, the change of CMV serology tests and qPCR assays, and the different cutoffs used for preemptive CMV treatment initiation during the study period [5▪▪,14,15]. Although awaiting additional reports from those data (manuscript under preparation), those are the only data reporting on the clinical significance of pretransplant recipient CMV IgG titers on posttransplant CMV DNAemia in the letermovir era.

Additional data from an elaborate discovery and validation cohort study were recently presented at the 50th annual meeting of the European Bone Marrow Transplant (EBMT) Society by the Seattle group [8]. Pretransplant sera from 478 allogeneic HCT D−R+ patients between 2007 and 2017, before the introduction of letermovir in clinical practice, were tested for CMV-specific humoral immunity by display immunoprecipitation and sequencing (VirScan). By analogy to CMV IgG titers, the antigen-antibody strength was assessed by measuring both CMV epitope hits (mean epitope binding signal, EBS; for all 478 patients) and CMV neutralizing antibodies (for 174 patients). Higher pretransplant CMV-specific humoral immunity titers were associated with higher cumulative incidence of CMV DNAemia (any and ≥150 IU/ml) during the first 100 days posttransplant in both the discovery and validation cohorts.

FUTURE PERSPECTIVES

The last couple of years have shifted the attention of our scientific community to CMV humoral immunity and CMI. Recent retrospective and prospective data from both allogeneic HCT and SOT recipients have suggested that CMV CMI may play an important role in controlling CMV reactivation posttransplant [16–18]. The clinical implication of those findings remains to be defined. The data on the clinical significance of pretransplant recipient CMV humoral immunity on posttransplant CMV replication presented in this article come to complement the existing body of literature and add one more piece to the complicated puzzle of CMV infection posttransplant. Those observations may potentially lead to future interventional studies, to further assess whether and how a combination of humoral and cellular CMV immunity could be incorporated in predictive models and algorithms to identify and potentially stratify patients to different primary CMV prophylactic strategies and duration.

CONCLUSION

The recent years have seen the reevaluation of pretransplant CMV serology, both as a mean to accurately define and identify truly CMV R+ allogeneic HCT recipients, but also as a potential predictor of posttransplant CMV replication. More data are urgently needed to better understand and describe the underlying pathophysiology between CMV IgG titer and CMV reactivation, in order to identify how quantified CMV IgG titer could be further incorporated in everyday clinical practice.

Acknowledgements

The authors would like to thank their patients and all personnel in the bone marrow transplant unit.

Financial support and sponsorship

There was no financial support or sponsorship for this article.

Conflicts of interest

L.R.: no conflicts of interest.

D.N. reports research support from MSD and Pfizer and consulting fees from MSD, Pfizer, Basilea, Takeda, and Gilead.

REFERENCES AND RECOMMENDED READING

Papers of particular interest, published within the annual period of review, have been highlighted as:

▪ of special interest

▪▪ of outstanding interest

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