Preemptive therapy at CMV reactivation has diminished post-transplant CMV mortality. Furthermore, recent studies suggest a favorable ‘virus-versus-leukemia’ effect from reactivating CMV, reducing relapse of AML after SCT. We studied the relationship of CMV reactivation with leukemic relapse in 110 patients with CML receiving HLA-identical sibling SCT between 1993 and 2008. Of these, 79 (72%) were in chronic phase, 5 in second chronic phase, 17 in accelerated phase and 9 in blast phase. A total of 97 patients (88%) received a myeloablative conditioning regimen, 97 received 4-log ex vivo T cell-depleted grafts and 13 received T-replete grafts. CMV reactivation before day 100 was observed in 72 patients (65.5%). At a median follow-up of 6.2 years, CMV reactivation < day 100 as a time-dependent covariate was an independent factor associated with decreased relapse. We conclude that CMV reactivation may contribute to a beneficial GVL effect in CML transplant recipients.
Reactivation of CMV is a frequent complication after allo-SCT but CMV-related mortality has been significantly diminished since the introduction of preemptive therapy for reactivating virus.1, 2, 3 Despite impressive reductions in CMV mortality, CMV reactivation and subsequent antiviral therapy are still regarded as harmful because of associations with increased risk from acute GVHD, myelosuppression and immunosuppression.4, 5, 6, 7 These concerns were challenged by recent studies in the era of CMV preemptive therapy, which suggested that early CMV reactivation after allo-SCT was independently associated with substantial reductions in leukemia relapse in adult patients with AML8 and pediatric patients with acute leukemias.9 These intriguing observations of a reduction in relapse have promoted a reassessment of the impact of CMV reactivation on transplant outcome. Although the protective effect of CMV reactivation on relapse appears robust, the mechanism underlying this effect remains unclear. It has been proposed that CMV reactivation initiates a T-cell or natural killer (NK) cell attack on leukemic progenitors either because they harbor the latent CMV virus or because CMV activates cytotoxic lymphocytes that kill bystander leukemic cells. This would imply that the protective effect of CMV reactivation on relapse is time dependent—observed only when lymphocyte function is adequate to react to the virus but limited by the return of sufficient immune function to suppress further viral reactivation. This usually occurs within 3 to 4 months post SCT. Whether the CMV effect on relapse is restricted to specific hematologic malignancies is not known. To further evaluate the impact of CMV reactivation after allo-SCT on myeloid malignancies other than AML, we analyzed the relationship of CMV reactivation with relapse in a cohort of consecutive patients with CML receiving HLA-identical sibling allo-SCT.
Materials and methods
Between September 1993 and August 2008, 110 consecutive patients with Ph chromosome-positive CML underwent allo-SCT from a 6/6 HLA-identical sibling in successive National Heart, Lung and Blood Institute (NHLBI) institutional review board-approved protocols (93-H-0212, 97-H-0202, 97-H-0099, 00-H-0001, 01-H-0010, 02-H-0111, 03-H-0192, 04-H-0112, 06-H-0248 and 07-H-0136). The study covered the period in which scheduled CMV screening and preemptive anti-CMV treatment was routinely performed. Written informed consent, consistent with the Helsinki Declaration, was obtained from all patients and donors.
Transplant conditioning regimen and ex vivo T-cell depletion
A total of 97 patients (88%) received a myeloablative conditioning regimen that included CY 120 mg/kg±fludarabine 125 mg/m2 and TBI from 1200 to1360 Gy; 13 patients (12%) received a non-myeloablative conditioning regimen consisting of CY 120 mg/kg and fludarabine 125 mg/m2. In addition, 27 patients (24.5%) received BM and 83 patients (75.5%) received G-CSF-mobilized peripheral blood as the stem cell source. A total of 13 patients (12%) received a T lymphocyte-replete graft and 97 (88%) received a 4-log10 ex vivo T lymphocyte-depleted graft. T-cell depletion was accomplished by the CellPro system (CellPro Inc., Bothel, WA, USA) before 1999, Isolex 300i (Nexell Therapeutics Inc., Irvine, CA, USA) from 1999 to 2006 and thereafter positive selection of CD34+ cells using the Miltenyi CliniMacs system (Miltenyi Biotec Inc., Auburn, CA, USA). CsA was used throughout for GVHD prophylaxis, with varied dose according to protocol.10 In all, 55 patients received standard-dose CsA (target plasma level, 200–400 μg/ml) and 55 received low-dose CsA (target plasma level, 100–200 μg/ml). In addition to CsA, MTX and/or mycophenolate mofetil were used in T-replete graft protocols. A total of 63 patients (57%) received a protocol scheduled infusion of donor lymphocytes before day 100.
CMV reactivation was defined as either CMV pp65 antigenemia, at least 1 positive cell per 400 000 WBCs or detection of more than 250 copies/ml of CMV DNA by quantitative real-time PCR (Q-CMV PCR). CMV disease required the identification of CMV in biopsy specimens. CMV retinitis was diagnosed on retinal examination by an experienced ophthalmologist.11 CMV antigenemia was performed on whole blood routinely at least weekly until day 100 after SCT and thereafter only if clinically indicated. In the absence of comprehensive CMV monitoring data, we categorized five patients who developed their first CMV reactivation after day 100 as CMV non-reactivators. To explore temporal associations between CMV reactivation and relapse reactivation before 30 days or 31–100 days after transplantation were separately analyzed. As a surrogate for immune competence the frequency of reactivation was subcategorized into one time reactivation (robust immunity) or reactivation at multiple time points (weaker immunity).
CMV monitoring and management
Before March 2005, CMV pp65 blood antigenemia was monitored weekly in buffy coat until at least day 100 after transplantation. Weekly or twice weekly Q-CMV PCR in whole blood was used for monitoring thereafter.12 All patient received acyclovir as antiviral prophylaxis. CMV reactivation was preemptively treated with either i.v. ganciclovir or oral valganciclovir. Foscarnet was substituted in the patients with cytopenias or ganciclovir-refractory cases. An initial antiviral induction dose was continued for 10–14 days followed by maintenance dose until 2 consecutive negative surveillance results were recorded.
Disease monitoring and management of relapse
Patients were monitored for molecular evidence of leukemia by real time quantitative PCR (RQ-PCR) for BCR-ABL on blood samples collected at least every 3 months up to 1 year post transplant, then at least annually afterward. Relapse included: (1) molecular relapse, defined as a detectable BCR/ABL transcript level by RQ-PCR on more than two consecutive occasions, requiring salvage treatments such as DLI, tyrosine kinase inhibitor or second hematopoietic SCT; and (2) any hematological relapse as defined by Center for International Bone and Marrow Transplantation Registry (CIBMTR) criteria.13
OS was defined as the time to transplantation until death from any cause. Relapse-free survival (RFS) was defined as the survival without positive RQ-PCR, cytogenetic or hematological evidence of CML relapse. Multivariate analysis was performed with Cox proportional hazard models for OS, RFS and relapse. CMV reactivation was included as a time-dependent covariate.14 Other variables included in the analysis were early CMV reactivation before day 30, CMV seronegative/seropositive recipients, age (older or younger than 40 years), disease status at transplantation (chronic phase vs advanced phase), conditioning regimen (myeloablative vs nonmyeloablative), graft manipulation (T deplete vs T replete), donor recipient sex match (female to male vs others), acute GVHD (grade 0–1 vs grade 2–4), donor/recipient CMV serology, scheduled add-back DLI, stem cell source (BM vs peripheral blood) and chronic GVHD in 100-day survivors. Statistical analysis was performed using SPSS (IBM, version 17.0, Chicago, IL, USA). Variables that attained a P-value of ⩽0.05 were held in the final multivariate models using stepwise backward selection method.
Median time to transplantation was 22 months and 79 patients (72%) were in chronic phase at transplantation (Table 1). Of the patients, 93 (84.5%) had never received tyrosine kinase inhibitors before transplantation because of either pre-tyrosine kinase inhibitor era or socioeconomic reasons. At a median follow-up of 6.2 years, 54 patients (49%) relapsed (32 molecular and 22 hematological relapses). A total of 44 patients (40%) died, 16 from relapse and 28 from nonrelapse causes, including 4 who died from CMV-related disease (pneumonitis). Also, 12 patients died before 100 days post transplant. The 10-year OS was 57%, the 10-year RFS was 26% and the 10-year current RFS was 57%.
Pretransplantation CMV serology, CMV reactivation and CMV disease
Of the 110 patients studied, 97 (88%) were CMV seropositive before transplant and 84 (76%) received their graft from CMV seropositive donors(Table 2). CMV reactivation before day 100 post transplant (CMV reactivation < day 100) was observed in 72 of these patients (65.5%), with a median time from transplantation to CMV reactivation of 37 days; 65 (90%) with CMV antigenemia or positive Q-CMV PCR and 7 with CMV disease (5 pneumonitis, 1 retinitis and 1 colitis). In addition, 28 patients (39%) had only one-time CMV reactivation and 44 patients (61%) experienced multiple CMV reactivations. All except 2 patients had the first CMV reactivations occurring before the relapse at a median of 723 days (35–3684) from first reactivation to relapse.
Relapse, RFS, OS and CMV reactivation
In multivariate analysis, time-dependent CMV reactivation < day 100 emerged as a significant independent factor for relapse (hazard ratio=0.533; 95% confidence interval, 0.288–0.986, P=0.045) in addition to stem cell source and pretransplant disease status (Table 3). In multivariate analysis, CMV donor–recipient serostatus, frequency of CMV reactivation (one-time vs multiple-time reactivators), CMV reactivation < day 30 were not significantly associated with relapse. OS and RFS were not significantly associated with CMV reactivation.
In a subgroup analysis of day 100 survivors (n=98), time-dependent CMV reactivation < day 100 remained as an independent factor for relapse (P=0.038) in multivariate analysis after including chronic GVHD as a covariate. In a subgroup analysis of pretransplant CMV seropositive recipients (n=97), time-dependent CMV reactivation < day 100 was not an independent factor for relapse in multivariate analysis.
We have previously shown that CML patients undergoing ex vivo T-depleted allo-SCT have a high rate of molecular relapse.15 Our observation that post-transplant CMV reactivation was one of the independent factors associated with less relapse in multivariate analysis in patients with CML extends the finding by Elmaagacli et al.8 who reported a beneficial impact of CMV reactivation on relapse in AML SCT recipients, independent of GVHD, and coined the term ‘virus-versus leukemia effect’.
In contrast to the AML study, we found no significant benefit of CMV reactivation on OS in CML patients. This would be anticipated as many relapsed CML patients were successfully salvaged by tyrosine kinase inhibitors and/or DLIs.13, 15 The quality of the immune response to CMV or EBV has been previously shown to serve as a surrogate for donor T cell-mediated antileukemia effects.16, 17, 18 Thomson et al.19 recently reported no significant association of AML relapse and CMV reactivation in alemtuzumab-based T-deplete allo-SCT. Consistent with a prompt immune recovery in T-replete grafts, the impact of CMV reactivation on relapse in our cohort was notable in this small subset, with all T-replete graft recipients not reactivating CMV experiencing relapse of disease (data not shown). Taken together, the results suggest that rather than a ‘virus-versus leukemia’ effect, CMV reactivation requires a T- or natural killer (NK)-cell immune response to enhance a GVL effect.
Mechanisms proposing how CMV reactivation and GVL may be linked still lack experimental confirmation.20 Myeloid leukemias may serve as a reservoir for CMV, and virus reactivation may increase leukemic immunogenicity, making them better targets for leukemia (or viral)-specific T cells or NK cells.21, 22 Another possibility is that CMV reactivation induces increased NK cell-mediated GVL: CMV reactivation can promote NK cell maturation in the early post-transplant period,23, 24 and early post-transplant NK cell recovery is favorable for rapid elimination of CML.25 Thus, CMV reactivation in BM or secondary lymphoid tissues may both stimulate NK cell reconstitution and sensitize leukemia cells to NK cell cytotoxicity. Alternatively, dynamic changes of cytokines in the BM milieu after CMV reactivation may boost antileukemia immunity through a bystander effect. Because of potential constraints of this multivariate analysis of retrospective data in a heterogeneous cohort of patients, we explored other reasons for the supposed relationship between CMV reactivation and relapse. Because the study covers more than a decade of transplant practice, it is also possible that unperceived differences in management and CMV monitoring may have taken place. Another issue is the interaction of competing variables with our findings. Because 12 patients died before day 100, we reanalyzed the 98 survivors at day 100 and still found the same correlations in multivariate analysis. Nevertheless, we cannot determine how much CMV reactivation is simply a surrogate for immunological factors that directly affect relapse. In particular, it would have been of interest to extend an earlier study of NK cell recovery in 20 patients in this cohort to identify a potential relationship between lymphocyte recovery, CMV reactivation and relapse.25
In vitro experiments may help substantiate a new paradigm of antileukemia effects in relation to viral immunobiology. Ultimately, such insights might modify transplant practice including donor selection strategies and optimization of post-transplantation immunosuppressant schedules to target early CMV reactivation. Our retrospective analysis and recent reports are limited to myeloid malignancies and HLA-matched donor transplants.
Finally, a role for CMV reactivation in relapse prevention deserves evaluation in other hematologic malignancies, especially lymphoid disease and also in alternative donor SCT with cord blood where the potential benefit of viral reactivation might be offset by mortality associated with a high incidence of CMV reactivation-refractory preemptive therapy.26
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This research was supported by the Intramural Research Program of the National institutes of Health, at the National Heart, Lung, and Blood Institute.
The authors declare no conflict of interest.
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Ito, S., Pophali, P., CO, W. et al. CMV reactivation is associated with a lower incidence of relapse after allo-SCT for CML. Bone Marrow Transplant 48, 1313–1316 (2013). https://doi.org/10.1038/bmt.2013.49
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