Abstract
Cytomegalovirus (CMV) reactivation after allogeneic hematopoietic cell transplant (allo-HCT) has been associated with reduced risk of relapse in patients with acute myeloid leukemia (AML). However the influence of the conditioning regimen on this protective effect of CMV reactivation after allo-HCT is relatively unexplored. To address this, we evaluated the risk of relapse in 264 AML patients who received T cell replete, 6/6 HLA matched sibling or 10/10 HLA matched unrelated donor transplantation at a single institution between 2006 and 2011. Out of these 264 patients, 206 received myeloablative (MA) and 58 received reduced intensity conditioning (RIC) regimens. CMV reactivation was observed in 88 patients with MA conditioning and 37 patients with RIC. At a median follow up of 299 days, CMV reactivation was associated with significantly lower risk of relapse in patients who received MA conditioning both in univariate (P= .01) and multivariate analyses (hazard ratio of 0.5246, P= .006), however CMV reactivation did not significantly affect the risk of relapse in our RIC cohort. These results confirm the protective effect of CMV reactivation on relapse in AML patients after allo-HCT reported by previous studies, however they suggest that this protective effect of CMV reactivation on relapse is influenced by the conditioning regimen used with the transplant.
Introduction
Cytomegalovirus (CMV) is a double stranded DNA β herpes virus that is generally of no major clinical significance in healthy immunocompetent hosts but is responsible for significant morbidity and mortality in immunocompromised patients1,2. In patients with allogeneic hematopoietic cell transplant (allo-HCT), the incidence of CMV disease has significantly reduced due to early detection of CMV reactivation and use of preemptive antiviral therapy. In spite of this, CMV reactivation remains a significant cause for morbidity and mortality among allo-HCT patients3–5. Interestingly in a recent study by Elmaagacli et al, early CMV pp65 antigenemia after allo-HCT was associated with reduced risk of relapse in AML patients6. This study included a relatively homogeneous population who underwent fully matched allo-HCT with myeloablative (MA) conditioning. In a large cohort of patients, using CMV pp65 antigenemia monitoring, Green et al found a modest protection against relapse in AML patients after allo-HCT, which included both MA and reduced intensity conditioning (RIC) patients, but the cohorts were analyzed together with no subgroup analysis7. Currently the influence of conditioning regimen on this protective effect of CMV reactivation on the risk of relapse is relatively unexplored. Quantitative CMV polymerase chain reaction (qPCR) is a more sensitive assay compared to pp65 antigenemia for CMV detection and has been shown to assist in early detection of CMV reactivation after allo-HCT leading to prompt preemptive treatment of CMV viremia3,8,9. Whether implementing CMV qPCR instead of PP65 antigenemia assay alters this association of reduced relapse risk with CMV reactivation after allo-HCT in AML patients is also currently not known.
To address the above questions, we retrospectively analyzed 264 AML patients who received T cell replete, 6/6 HLA matched sibling or 10/10 HLA matched unrelated donor transplantation at a single institution between 2006 and 2011.
Patients and Methods
Study Population
The study included a total of 382 consecutive AML patients who underwent allo-HCT at Washington University Medical Center at St Louis, between January 2006 and December 2011. This study was approved by Institutional review board (IRB) of Washington University School of Medicine, St Louis. Patient demographics and transplant characteristics were prospectively entered into Washington University School of Medicine, Blood and Marrow transplant database. 264 out of these 382 patients were selected for the analysis based on following eligibility criteria: (1) 10 out of 10 match at human leucocyte antigen (HLA) loci A, B, C, DRB1 and DQB1 by high resolution genotyping in unrelated transplantation10 and by low resolution11 in related donor transplantation (2) use of unmodified donor stem cells (3) no use of prophylactic DLI during the post transplantation course among patients without leukemic relapse (4) bone marrow biopsy done within 30 days prior to transplant to determine the disease status at the time of transplantation, and (5) recipients of a second transplant were excluded from the study group as prior transplant.
The type of conditioning regimen patients received was classified according to consensus definition of conditioning regimen intensity12. For our study reduced intensity and non-myeloablative regimens were grouped together under RIC cohort.
Definitions
Monitoring for CMV reactivation was done through quantitative (real time) CMV PCR assay. The theoretical lower limit of detection of the assay is 200 genome copies per ml of blood (c/ml) and considered negative/undetectable below this limit. The assay is accurate for quantitation above 2,000 c/ml and any value greater than 200 c/ml but less than 2000 c/ml was defined as positive but not quantifiable. A CMV viral load greater than 2000 c/ml was considered positive with a quantifiable viral load. CMV viral load greater than 200 c/ml was considered as CMV reactivation and this value was used for analyzing its influence on relapse risk in this study.
Acute GVHD (aGvHD) was diagnosed clinically based on signs and symptoms and graded according to accepted criteria13. Chronic GVHD (cGvHD) was graded in accordance with NIH consensus criteria for diagnosis and grading14.
Etiology of AML was classified into de novo AML without antecedent diagnosis of bone marrow disorders such as myelodysplastic syndrome (MDS) or myeloproliferative disorder (MPD), secondary AML (sAML) occurring from underlying bone marrow disorders such as MDS or MPD and therapy related AML (tAML) occurring as a result of prior exposure to chemotherapy and or radiation therapy as per WHO classification15, 16. AML was classified into good, intermediate and poor prognostic groups based on cytogenetic and molecular abnormalities 17,18,19. Disease status at the time of transplant was classified as complete remission-1 (CR-1), complete remission (CR-2), active disease and ‘Other’ based on the bone marrow biopsy done within 30 days prior to the transplantation. 20,21 ‘Other’ group included patients who achieved remission for the third time or beyond. Patients with persistent disease at the time of transplant were classified as active disease.
CMV Viremia Monitoring and Treatment
qPCR testing for CMV reactivation after allo-HCT was done at least twice weekly for all inpatients and once weekly in the outpatient setting. CMV viremia when detected and quantifiable was treated with IV ganciclovir or oral valganciclovir for 14 days and if follow up CMV qPCR showed improvement in the viral load then continued on maintenance dose until 2 consecutive qPCR results were negative22,23 At that point antiviral treatment was changed back to either acyclovir or valacyclovir.
Post-Transplant Disease Monitoring
Patients underwent bone marrow biopsies after allo-HCT at 30 days, 100 days and then every 6 months or earlier if there were any abnormal findings noted on their peripheral blood suspicious for relapse. Relapse was defined per accepted criteria16. Morphologic relapse was the primary outcome for patients transplanted in CR and progression of disease for patients transplanted with active disease. Engraftment of the donor cells was determined by southern blotting test for short tandem repeats (STRs) from peripheral blood mononuclear cells and or bone marrow samples24. All patients were followed for CMV reactivation data till the patients relapsed or for those without relapse, till death or the last clinic visit. Complete donor engraftment was defined by the presence of less than 5% of the recipient cells obtained from bone marrow sample STRs. Recipient cell percentage greater than 5% was defined as mixed chimerism. Extramedullary disease or relapse was defined by, by presence of blasts in tissue biopsy or by the presence of morphologically or phenotypically positive blasts in the CSF.
Study Endpoints and Statistical Analyses
The primary study endpoint was cumulative incidence of relapse while secondary endpoints included overall survival (OS), relapse free survival (RFS) and NRM. Cumulative incidence of relapse was compared between groups with and without CMV reactivation for statistical significance. Cumulative incidence of relapse was estimated treating death in remission as competing risk event and cumulative incidence of NRM was estimated treating death in relapse as competing risk event. OS was defined as the time from allo-HCT to death from any cause or last follow-up. RFS was defined as the time from allo-HCT to relapse or death without relapse whichever occurred first.
Patient-, disease- and treatment-related variables for the two study groups were compared using the chi-square statistic or Fisher's Exact test for categorical and the Kruskal-Wallis test for continuous variables. The cumulative incidence plots were generated by SAS macro %CIF and Gray's test was used to test equality of cumulative incidence function between two study groups. Kaplan-Meier (KM) curves for OS were generated that provide unadjusted survival estimates between study groups. Differences between study groups were determined by log-rank tests. All statistical tests were two-sided using a α = .05 level of significance. SAS Version 9.3 (Cary, NC) was used to perform all statistical analyses.
Univariate and multivariable proportional subdistribution hazards models using the Fine and Gray approach25 were considered to evaluate the variables for relapse, treating death in remission as competing event. The variables included CMV reactivation status (yes vs. no), cGVHD (yes vs. no), donor/recipient CMV status, use of radiation therapy in conditioning regimen, use of ATG in conditioning regimen, CMV reactivation within 100 days after transplant (yes vs. no). A forward stepwise model selection approach was performed to identify all significant risk factors. Factors significant at a 10% level were kept in the final model. CMV reactivation was forced into the multivariate model. The cmprsk package in R was used for this analysis. Moreover, Kolmogorov-Smirnov test and Cramer von Mises test in the timereg's comp.risk () function was used to test time invariant effect for each interested variables.
Results
Patient Characteristics
Based on CMV reactivation status we divided our entire patient cohort into two groups. Patient, disease, and transplant characteristics of these cohorts with and without CMV reactivation are summarized in Table 1. A total of 125 patients had CMV reactivation, out of which 100 had CMV reactivation within the first 100 days of transplantation. Median time for CMV reactivation was 33 days (range: 3-666). A total of 100 patients developed Grade II-IV aGvHD, 44 (44%) in the CMV reactivation cohort and 56 (56%) in the non-CMV reactivation cohort and this difference was not statistically significant (P= .446). Similarly, a total of 89 patients developed cGvHD, 40 (45%) patients in the CMV reactivation cohort and 49 (55%) in the non-CMV reactivation cohort and this difference was also not statistically significant (P= .602). 108 patients underwent matched related donor (MRD) transplant and 156 patients underwent matched unrelated donor transplant (MUD) and this difference in the type of transplants (MRD vs. MUD) between the cohorts with and without CMV reactivation was not significant (P= .803). Similarly there was no significant difference in distribution by disease etiology, disease prognosis, or by disease status at transplant. Distribution of gender was also similar across both the study groups.
Table 1. Patient, Donor and Transplant Characteristics.
| All patients | Patients with CMV reactivation | Patients without CMV reactivation | p-value | |
|---|---|---|---|---|
|
| ||||
| Patient number (%) | 264(100) | 125 | 139 | - |
|
| ||||
| Median patient age (range) | - | 56 (23-73) | 51 (17-68) | 0.005 |
|
| ||||
| Patient sex (%)- | .805 | |||
| Female | 125(47) | 58 (46) | 67 (48) | |
| Male | 139(53) | 67 (54) | 72 (52) | |
|
| ||||
| Donor sex (%)– | .696 | |||
| Female | 88(33) | 40 (32) | 48 (35) | |
| Male | 158(67) | 85 (68) | 91 (65) | |
|
| ||||
| Donor/patient sex | .397 | |||
| Female/Male | 40(15) | 15(12) | 25(18) | |
| Other | 224(85) | 110(88) | 114(82) | |
|
| ||||
| Donor/patient CMV status (%) | <.0001 | |||
| Negative/Negative | 94 (36) | 11 (9) | 83 (60) | |
| Negative/Positive | 73 (28) | 59 (48) | 14 (10) | |
| Positive/Negative | 25 (9) | 8 (7) | 17(12) | |
| Positive/Positive | 68 (26) | 44 (36) | 24(17) | |
| Unknown | 4 (1) | |||
|
| ||||
| Disease etiology | .215 | |||
| de novo | 201 (76) | 96(77) | 105 (75) | |
| Secondary | 42 (16) | 16(13) | 26 (19) | |
| Therapy related | 21 (8) | 13(10) | 8 (6) | |
|
| ||||
| Transplant type (%) | .803 | |||
| MRD | 108 (41) | 50 (40) | 58 (42) | |
| MUD | 156 (59) | 75 (60) | 81 (58) | |
|
| ||||
| Conditioning regimen (%) | ||||
| Myeloablative | 206 (78) | 88 (70) | 118 (85) | .005 |
| RIC | 58 (22) | 37 (30) | 21 (15) | |
|
| ||||
| Disease classification by cytogenetics (%) | .089 | |||
| Favorable | 25 (9) | 17 (14) | 8 (8) | |
| Intermediate | 157 (60) | 70 (57) | 87 (46) | |
| Poor | 78 (30) | 35 (29) | 43 (44) | |
| Unknown | 4 (1) | |||
|
| ||||
| Disease status at transplant (%) | .339 | |||
| CR1 | 135 (51) | 69 (55) | 66 (48) | |
| CR2 | 58 (22) | 29 (23) | 29 (21) | |
| Active disease | 50 (19) | 29 (15) | 31 (22) | |
| Other | 21 (8) | 8 (6) | 13 (9) | |
|
| ||||
| aGvHD (%) | .446 | |||
| Gr 0-1 | 164 (63) | 81 (65) | 83 (60) | |
| Gr 2-4 | 100 (37) | 44 (35) | 56 (40) | |
|
| ||||
| cGvHD (%) | .602 | |||
| No | 172 (65) | 84 (68) | 88 (64) | |
| Yes | 89 (34) | 40 (32) | 49 (36) | |
| Unknown | 3 (1) | |||
|
| ||||
| ATG regimen (%) | .009 | |||
| Yes | 46 (17) | 30 (24) | 16 (12) | |
| no | 218 (83) | 95 (76) | 123 (88) | |
|
| ||||
| Stem cell source | 0.823 | |||
| Peripheral blood | 240 (91) | 115 (92) | 125 (91) | |
| Bone marrow | 23 (9) | 10 (8) | 13 (9) | |
|
| ||||
| Immune prophylaxis | .023 | |||
| MTX, MMF, Tacrolimus | 30(11) | 21 (17) | 9 (6) | |
| MTX, Tacrolimus | 215(81) | 97 (78) | 118 (85) | |
| Other* | 19(7) | 7 (5) | 12 (9) | |
MTX- Methotrexate, MMF- mycophenolate mofetil, ATG- anti-thymocyte globulin
- Other- includes cyclosporine, MTX, Sirolimus and tacrolimus, MRD- matched related donor, MUD- matched unrelated donor
There were few significant differences between these two groups. Patients in the CMV reactivation group were older and more likely had a patient/ donor combination of CMV seropositive/ seronegative status. Patients who had RIC allo-HCT and anti-thymocyte globulin (ATG) as part of their conditioning regimen were more likely to have CMV reactivation. 88 of 206 patients who underwent MA allo-HCT had CMV reactivation, whereas 37 patients out of 58 patients who underwent RIC allo-HCT had CMV reactivation (P= .005). 30 out of 46 patients with ATG in their conditioning regimen had CMV reactivation compared to 95 out of 218 patients without ATG in their conditioning regimen (P= .001).
The patient characteristics of our RIC and MA cohorts are described in Table 2.
Table 2. Patient, Donor and Transplant Characteristics by Intensity of Conditioning Regimen.
| All patients | MA | RIC | p-value | |
|---|---|---|---|---|
|
| ||||
| Patient number (%) | 264(100) | 125 | 139 | - |
|
| ||||
| Median patient age (range) | 50(17-68) | 62(21-73) | <.0001 | |
|
| ||||
| Patient sex (%) | 1.0 | |||
| Female | 125(47) | 98(48) | 27(47) | |
| Male | 139(53) | 108(52) | 31(53) | |
|
| ||||
| Donor sex (%)– | .432 | |||
| Female | 88(33) | 66(68) | 22(38) | |
| Male | 176(67) | 140(32) | 36(62) | |
|
| ||||
| Donor/Patient sex | .755 | |||
| Female/Male | 40(15) | 29(14) | 11(19) | |
| Other | 224(85) | 177(86) | 47(81) | |
|
| ||||
| Donor/Patient CMV status (%) | .0501 | |||
| Negative/Negative | 94(36) | 82(40) | 12(21) | |
| Negative/Positive | 73(28) | 54(27) | 19(34) | |
| Positive/Negative | 25(9) | 17(8) | 8(14) | |
| Positive/Positive | 68(26) | 51(25) | 17(30) | |
| Unknown | 4(1) | |||
|
| ||||
| Disease etiology | .443 | |||
| de novo | 201(76) | 158(77) | 43(74) | |
| Secondary | 42(16) | 30(14) | 12(12) | |
| Therapy related | 21(8) | 18(9) | 3(5) | |
|
| ||||
| Transplant type (%) | .291 | |||
| MRD | 108(41) | 88(43) | 20(34) | |
| MUD | 156(59) | 118(57) | 38(66) | |
|
| ||||
| CMV reactivation | .005 | |||
| Yes | 206(78) | 88(43) | 37(64) | |
| No | 58(22) | 118(57) | 21(36) | |
|
| ||||
| Disease classification by cytogenetics (%) | .154 | |||
| Favorable | 25(9) | 23(11) | 2(3) | |
| Intermediate | 157(60) | 118(58) | 39(67) | |
| Poor | 78(30) | 61(30) | 17(29) | |
| Unknown | 4(1) | |||
|
| ||||
| Disease status at transplant (%) | .012 | |||
| CR1 | 135(51) | 100(48) | 35(60) | |
| CR2 | 43(22) | 43(21) | 15(26) | |
| Active disease | 50(19) | 47(23) | 3(5) | |
| Other | 21(8) | 16(8) | 5(9) | |
|
| ||||
| Acute GvHD (%) | .015 | |||
| Gr 0-1 | 164(63) | 120(58) | 44(76) | |
| Gr 2-4 | 100(37) | 86(42) | 14(24) | |
|
| ||||
| Chronic GvHD (%) | .273 | |||
| No | 172(65) | 130(64) | 42(72) | |
| Yes | 89(89) | 73(36) | 16(28) | |
| Unknown | 3(1) | |||
|
| ||||
| ATG regimen (%) | <.0001 | |||
| Yes | 46(17) | 2(1) | 44(76) | |
| No | 218(83) | 204(99) | 14(24) | |
|
| ||||
| Stem cell source | .429 | |||
| Peripheral blood | 240(91) | 185(90) | 55(95) | |
| Bone marrow | 23(9) | 20(10) | 3(5) | |
| Missing information | 1 | |||
|
| ||||
| Immune prophylaxis | <.0001 | |||
| MTX, MMF, Tacrolimus | 30(11) | 7(3) | 23(40) | |
| MTX, Tacrolimus | 215(81) | 181(88) | 34(58) | |
| Other* | 19(7) | 18(9) | 1(2) | |
MA- Myeloablative regimen, RIC- Reduced intensity conditioning regimen, MTX- Methotrexate, MMF-mycophenolate mofetil, ATG- Anti-thymocyte globulin,
-other- includes cyclosporine, MTX, Sirolimus and Tacrolimus, MRD- matched related donor, MUD- matched unrelated donor
CMV reactivation and risk of relapse after allogeneic transplantation
Using highly sensitive qPCR method of CMV reactivation monitoring we evaluated the impact of CMV reactivation on the risk of relapse in patients who underwent allo-HCT. In our entire cohort of 264 patients median relapse free survival was 287 (range: 1-2423). 113 (46%) patients among the entire cohort relapsed at a median time of 140 days (range: 6-2041 days). Median relapse free survival was 290 days (range: 23-2423) in patients with CMV reactivation and 285 (range: 1-2366) in patients without CMV reactivation. The cumulative incidence of relapse among patients with CMV reactivation was 31.4%, 37.1% and 38.9% compared to 39%, 50% and 59% for patients without CMV reactivation at 1 year, 3 years and 6 years respectively (Figure 1). In the multivariable model CMV reactivation was still significantly associated with reduced risk of relapse (HR= .642, 95% CI .44-.94, P= .024) (Table 3). 37 patients relapsed within 100 days after their transplant out of which 27 patients relapsed before CMV reactivation.
Figure 1. Cumulative incidence of relapse stratified by CMV reactivation in all patients, MA patients and RIC patients.
Table 3. Variables Influencing Risk of Relapse on Multivariate Analysis.
| Parameter | Parameter Estimate | Standard Error | P value | Hazard Ratio | 95% Hazard Ratio Confidence Limits | |
|---|---|---|---|---|---|---|
| CMV (yes vs. no) | -0.4427 | 0.1965 | 0.0242 | 0.6423 | 0.4370 | 0.9440 |
| cGvHD (yes vs. no) | -1.1237 | 0.2243 | <.0001 | 0.3251 | 0.2094 | 0.5046 |
| XRTregimen (yes vs. no) | 0.4407 | 0.1948 | 0.0237 | 1.5538 | 1.0606 | 2.2763 |
CMV react- Post transplant CMV reactivation (yes vs. no), XRTregimen- use of radiation in conditioning regimen
Subgroup analysis was performed to assess whether there was any difference in relapse with CMV reactivation in our MA and RIC cohorts. In the MA cohort, 86 patients had relapsed by the end of the study. Median relapse free survival was 333 days (range: 23-2423 days) in subjects with CMV reactivation compared to 283 days (range: 1-2093 days) without CMV reactivation. Cumulative incidence of relapse was 28.6%, 33.7% and 33.7% at 1year, 3 years and 6 years respectively, in patients with CMV reactivation and 40% and 51.4% and 55.4% at 1 year, 3 years and 6 years respectively, in patients without CMV reactivation (Figure 1). This decreased risk of relapse with CMV reactivation in the MA cohort was significant as an independent factor in multivariate analysis (HR: .525; 95% CI: .331-.832, (P= .015) after controlling for cGvHD, use of ATG and radiation in the conditioning regimen (Table 4). In the same analysis, cGvHD was also associated with decreased risk of relapse (HR: .364, 95% CI: .220- .602, P< .0001) in our MA cohort.
Table 4. Variables Influencing Risk of Relapse on Multivariate Analysis in the MA Cohort.
| Parameter | Parameter Estimate | Standard Error | P value | Hazard Ratio | 95% Hazard Ratio Confidence Limits | |
|---|---|---|---|---|---|---|
| CMV (yes vs. no) | -0.6452 | 0.2353 | 0.0061 | 0.5246 | 0.3308 | 0.8319 |
| cGvHD (yes vs. no) | -1.0197 | 0.2458 | <.0001 | 0.3607 | 0.2228 | 0.5840 |
| XRTregimen (yes vs. no) | 0.5807 | 0.2231 | 0.0092 | 1.7873 | 1.1543 | 2.7674 |
| ATGregimen (yes vs. no) | 1.9601 | 0.2753 | <.0001 | 7.1002 | 4.1394 | 12.1787 |
CMV react- Post transplant CMV reactivation (yes vs. no), XRTregimen- use of radiation in conditioning regimen, ATGregimen- use of ATG in conditioning regimen
In contrast to these findings in our MA cohort, we did not find any significant difference in the risk of relapse with CMV reactivation in our RIC cohort. In RIC patients, the median relapse free survival was 162 days (range: 26-2157 days) in patients with CMV reactivation and 293 days (range: 16-2311 days) in those without CMV reactivation. The cumulative incidence of relapse in these patients did not differ with CMV reactivation (Figure 1).
Survival and mortality with CMV reactivation
As with cumulative incidence of relapse, we found improved RFS with CMV reactivation in the entire and MA cohorts but not in the RIC patients (Figure 2). However this improved RFS did not translate into improved OS in any of the patient cohorts (Figure 3). A trend towards higher NRM with CMV reactivation was observed in the entire cohort (both MA and RIC patients) but did not reach statistical significance in any of the cohorts (Figure 4).
Figure 2. RFS stratified by CMV reactivation in all patients, MA patients and RIC patients.
Figure 3. OS stratified by CMV reactivation in all patients, MA patients and RIC patients.
Figure 4. Cumulative incidence of NRM stratified by CMV reactivation in all patients, MA patients and RIC patients.
Discussion
Recently there has been several studies addressing the impact of CMV reactivation on risk of relapse after allo-HCT for myeloid malignancies6,26,27. Consistent with the previous studies we show, in our retrospective analysis, that CMV reactivation after allo-HCT leads to decreased relapse and improved RFS in AML patients. In contrast to other studies however we found that this protective effect for relapse to be significantly influenced by the transplant conditioning regimen and was restricted to those AML patients undergoing MA allo-HCT. Furthermore our study used qPCR based CMV monitoring in contrast to pp65 CMV antigenemia testing used in previous studies. qPCR is more sensitive than pp65 antigenemia based assays used in previous reports8,9. In a study by Schulenberg et al detection of CMV reactivation by qPCR had a median lead time of 11 days before detection by pp65 antigenemia and remained positive for a median of 16 days after testing negative for pp65 antigen9. This leads to early initiation and more prolonged antiviral treatment thereby potentially altering the impact of CMV reactivation on relapse and survival benefit. However here we show decreased relapse with CMV reactivation despite this potential lead-time for CMV treatment when using highly sensitive qPCR method of CMV monitoring in our allo-HCT patients. CMV positive serostatus of the donor and recipients was associated with reduced relapse rates in pediatric population who had undergone allo-HCTfor acute leukemia27. In a recent report by Elmagaacli et al, CMV reactivation in AML patients after allo-HCT was associated with decreased relapse and improved OS, however this study only included patients who received MA conditioning6. Similarly, Green et al observed decreased risk of relapse with CMV reactivation in a cohort of AML patients including both MA and RIC patients, that was more pronounced within the first 100 days after transplant and not stastically significant by 1 year7. Both these studies used pp65 antigenemia based monitoring for CMV reactivation. Using both pp65 antigenemia and qPCR based assays, Ito et al found decreased relapse with CMV reactivation after allo-HCT in CMV patients28. Further, in our patients this beneficial effect of CMV reactivation on relapse was sustained. Despite decreased risk of relapse and improved RFS we were unable to correlate improved OS with CMV reactivation which may be related to the increased NRM with CMV reactivation as we saw a clear trend toward increased NRM with CMV reactivation (although did not reach statistical significance) and this increased NRM with CMV reactivation was also reported by Green et al7. Based on these results we hypothesize that decreased relapse risk (and therefore improved RFS) and increased NRM compete to influence OS in patients with CMV reactivation after allo-HCT. This would explain the clear trend of decreased OS seen in our RIC cohort where the lack of this protective effect on relapse favors the balance toward poor OS (Figure 3).
The mechanism(s) behind decreased relapse with CMV reactivation allo-HCT are currently not known. Recently there has been significant interest in better understanding the impact of NK cell allo-reactivity on relapse especially in patients undergoing allo-HCT for myeloid malignancies 29,30,31. Foley et al found selective expansion of the NKG2C+CD57+ NK cells with CMV reactivation and these cells have enhanced anti-leukemia responses in vitro32. The enhanced anti-leukemia responses by NKG2C+CD57+ NK cells could potentially be responsible for enhanced NK cell mediated “graft versus leukemia” (GvL) effect in vivo, translating into less relapse in patients who reactivate CMV after allo-HCT. Furthermore, CMV reactivation increases leucocyte fixation antigen-3 (LFA-3) expression on blasts carrying CMV leading to enhanced NK cell mediated lysis of the blasts26,33. NK cells mediated GvL effect in general seems to be restricted to patients with myeloid malignancies and could be the reason why this CMV reactivation related decreased relapse has been observed in patients with myeloid malignancies29,30,6,7,28. Donor derived T cells could also be contributing to this ‘relapse protective’ effect from CMV reactivation. Leukemic blasts could harbor CMV and upon reactivation activated donor derived T cells specific to CMV could therefore be directly cytotoxic to these blasts expressing CMV antigens26. Thus the GvL effect is pronounced in the scenario where recipient is seropositive than in any other combination and this has been demonstrated in our study as well as in others7. The role of T cells in this enhanced GvL effect is underscored by the failure to demonstrate decreased relapse with CMV reactivation in a study where patients underwent T cell depleted grafts with an alemtuzumab-containing conditioning regimen34.
In our RIC cohort 44 out of 58 patients received ATG as part of their conditioning regimen. In vivo T cell depletion by ATG in this cohort may result in mitigating the enhanced GvL effect induced by CMV reactivation, therefore again underscoring the importance of graft derived T cells in mediating this effect. Further, host derived memory T cells can persist for up to 6 months in RIC patients and contribute towards immunity against CMV 35,36. Persistence of these host T cells could contribute to clearing of CMV upon its reactivation and thereby possibly preventing optimal donor T cell and NK cell activation. Persistence of the host lymphocytes early after allo-HCT in RIC patients could also compete for cytokines with donor derived T and NK cells and therefore impair with enhanced GvL effect seen in the context of CMV reactivation. The smaller RIC cohort could also be a factor for not observing relapse benefit. Further studies in a larger cohort of patients with RIC regimen with and without ATG would be prudent to better understand role of conditioning regimen in this relapse benefit from CMV reactivation.
Reactivation of CMV after allo-HCT is traditionally considered an adverse event needing aggressive therapy and most of the transplant centers monitor for CMV reactivation very closely and institute aggressive pre-emptive therapy upon evidence of CMV reactivation. Here we show that CMV reactivation after allo-HCT, especially in MA conditioning recipients is associated with decreased risk of relapse and improved RFS in AML patients. The mechanisms responsible for this enhanced GvL with CMV reactivation need to be further evaluated as better understanding could potentially lead to developing novel CMV antigen based vaccination strategies aimed at reducing relapse risk after allo-HCT in the future.
Acknowledgments
Authorship statement: S.M., PK.B., and R.R., designed the study design and interpreted data. S.M., PK.B, S.J.M and K.E.S-G prepared the dataset and drafted the manuscript. J.L. did the statistical analysis. S.M., PK.B., K.E.S-G., J.F.D., J.L., T.A.F., G.U., P.W., A.F.C., M.A.S., R.V., C.N.A., I.P., M.J. and R.R. interpreted data and critically reviewed the manuscript. All authors approved the final manuscript.
Footnotes
Financial disclosures: Authors have no financial disclosures.
Conflict of interest statement: There are no conflicts of interest to report.
Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
References
- 1.Rafailidis PI, Mourtzoukou EG, Varbobitis IC, Falagas ME. Severe cytomegalovirus infection in apparently immunocompetent patients: a systematic review. [Accessed May 25, 2013];Virol J. 2008 5:47. doi: 10.1186/1743-422X-5-47. Available at: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2289809&tool=pmcentrez&rendertype=abstract. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Boeckh M, Ljungman P. How we treat cytomegalovirus in hematopoietic cell transplant recipients. [Accessed May 25, 2013];Blood. 2009 113(23):5711–9. doi: 10.1182/blood-2008-10-143560. Available at: http://bloodjournal.hematologylibrary.org/content/113/23/5711.full. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Ljungman P, Hakki M, Boeckh M. Cytomegalovirus in hematopoietic stem cell transplant recipients. [Accessed May 25, 2013];Hematol Oncol Clin North Am. 2011 25(1):151–69. doi: 10.1016/j.hoc.2010.11.011. Available at: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3340426&tool=pmcentrez&rendertype=abstract. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Ljungman P, Perez-Bercoff L, Jonsson J, et al. Risk factors for the development of cytomegalovirus disease after allogeneic stem cell transplantation. [Accessed May 25, 2013];Haematologica. 2006 91(1):78–83. Available at: http://www.ncbi.nlm.nih.gov/pubmed/16434374. [PubMed] [Google Scholar]
- 5.Broers AE, van Der Holt R, van Esser JW, et al. Increased transplant-related morbidity and mortality in CMV-seropositive patients despite highly effective prevention of CMV disease after allogeneic T-cell-depleted stem cell transplantation. [Accessed May 25, 2013];Blood. 2000 95(7):2240–5. Available at: http://www.ncbi.nlm.nih.gov/pubmed/10733491. [PubMed] [Google Scholar]
- 6.Elmaagacli AH, Steckel NK, Koldehoff M, et al. Early human cytomegalovirus replication after transplantation is associated with a decreased relapse risk: evidence for a putative virus-versus-leukemia effect in acute myeloid leukemia patients. [Accessed May 25, 2013];Blood. 2011 118(5):1402–12. doi: 10.1182/blood-2010-08-304121. Available at: http://bloodjournal.hematologylibrary.org/content/118/5/1402.full. [DOI] [PubMed] [Google Scholar]
- 7.Green ML, Leisenring WM, Xie H, et al. CMV reactivation after allogeneic HCT and relapse risk: evidence for early protection in acute myeloid leukemia. [Accessed August 16, 2013];Blood. 2013 :1316–1324. doi: 10.1182/blood-2013-02-487074. Available at: http://www.ncbi.nlm.nih.gov/pubmed/23744585. [DOI] [PMC free article] [PubMed]
- 8.Nitsche A, Oswald O, Steuer N, et al. Quantitative real-time PCR compared with pp65 antigen detection for cytomegalovirus (CMV) in 1122 blood specimens from 77 patients after allogeneic stem cell transplantation: which test better predicts CMV disease development? [Accessed May 25, 2013];Clin Chem. 2003 49(10):1683–5. doi: 10.1373/49.10.1683. Available at: http://www.ncbi.nlm.nih.gov/pubmed/14500600. [DOI] [PubMed] [Google Scholar]
- 9.Schulenburg A, Rabitsch W, Loidolt H, Keil F, Mitterbauer M, Kalhs P. Infections post transplant CMV monitoring after peripheral blood stem cell and bone marrow transplantation by pp65 antigen and quantitative PCR. 2001 Jul;:765–768. doi: 10.1038/sj.bmt.1703227. Summary. [DOI] [PubMed] [Google Scholar]
- 10.Speiser DE, Tiercy JM, Rufer N, et al. High resolution HLA matching associated with decreased mortality after unrelated bone marrow transplantation. Blood. 1996;87(10):4455–62. Available at: http://www.ncbi.nlm.nih.gov/pubmed/8639808. [PubMed] [Google Scholar]
- 11.Ottinger HD, Ferencik S, Beelen DW, et al. Hematopoietic stem cell transplantation: contrasting the outcome of transplantations from HLA-identical siblings, partially HLA-mismatched related donors, and HLA-matched unrelated donors. [Accessed May 22, 2013];Blood. 2003 102(3):1131–7. doi: 10.1182/blood-2002-09-2866. Available at: http://www.ncbi.nlm.nih.gov/pubmed/12689945. [DOI] [PubMed] [Google Scholar]
- 12.Bacigalupo A, Ballen K, Rizzo D, et al. Defining the intensity of conditioning regimens: working definitions. Biol blood marrow Transplant J Am Soc Blood Marrow Transplant. 2009;15(12):1628–1633. doi: 10.1016/j.bbmt.2009.07.004. Available at: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2861656&tool=pmcentrez&rendertype=abstract. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Glucksberg H, Storb R, Fefer A, et al. Clinical manifestations of graft-versus-host disease in human recipients of marrow from HL-A-matched sibling donors. [Accessed May 22, 2013];Transplantation. 1974 18(4):295–304. doi: 10.1097/00007890-197410000-00001. Available at: http://www.ncbi.nlm.nih.gov/pubmed/4153799. [DOI] [PubMed] [Google Scholar]
- 14.Filipovich AH, Weisdorf D, Pavletic S, et al. National Institutes of Health consensus development project on criteria for clinical trials in chronic graft-versus-host disease: I. Diagnosis and staging working group report. [Accessed March 6, 2013];Biol Blood Marrow Transplant. 2005 11(12):945–56. doi: 10.1016/j.bbmt.2005.09.004. Available at: http://www.ncbi.nlm.nih.gov/pubmed/16338616. [DOI] [PubMed] [Google Scholar]
- 15.Vardiman JW, Harris NL, Brunning RD. The World Health Organization (WHO) classification of the myeloid neoplasms. [Accessed May 23, 2013];Blood. 2002 100(7):2292–302. doi: 10.1182/blood-2002-04-1199. Available at: http://bloodjournal.hematologylibrary.org/content/100/7/2292.full. [DOI] [PubMed] [Google Scholar]
- 16.Cheson BD, Bennett JM, Kopecky KJ, et al. Revised recommendations of the International Working Group for Diagnosis, Standardization of Response Criteria, Treatment Outcomes, and Reporting Standards for Therapeutic Trials in Acute Myeloid Leukemia. [Accessed May 23, 2013];J Clin Oncol. 2003 21(24):4642–9. doi: 10.1200/JCO.2003.04.036. Available at: http://www.ncbi.nlm.nih.gov/pubmed/14673054. [DOI] [PubMed] [Google Scholar]
- 17.Mrózek K, Heerema Na, Bloomfield CD. Cytogenetics in acute leukemia. [Accessed May 23, 2013];Blood Rev. 2004 18(2):115–36. doi: 10.1016/S0268-960X(03)00040-7. Available at: http://www.ncbi.nlm.nih.gov/pubmed/15010150. [DOI] [PubMed] [Google Scholar]
- 18.Slovak ML, Kopecky KJ, Cassileth PA, et al. Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia: a Southwest Oncology Group/Eastern Cooperative Oncology Group study. [Accessed May 23, 2013];Blood. 2000 96(13):4075–4083. Available at: http://bloodjournal.hematologylibrary.org/content/96/13/4075.full. [PubMed] [Google Scholar]
- 19.Grimwade D, Walker H, Oliver F, et al. The Importance of Diagnostic Cytogenetics on Outcome in AML: Analysis of 1,612 Patients Entered Into the MRC AML 10 Trial. [Accessed May 23, 2013];Blood. 1998 92(7):2322–2333. Available at: http://bloodjournal.hematologylibrary.org/content/92/7/2322.full. [PubMed] [Google Scholar]
- 20.Cornelissen JJ, van Putten WLJ, Verdonck LF, et al. Results of a HOVON/SAKK donor versus no-donor analysis of myeloablative HLA-identical sibling stem cell transplantation in first remission acute myeloid leukemia in young and middle-aged adults: benefits for whom? [Accessed May 23, 2013];Blood. 2007 109(9):3658–66. doi: 10.1182/blood-2006-06-025627. Available at: http://bloodjournal.hematologylibrary.org/content/109/9/3658.long. [DOI] [PubMed] [Google Scholar]
- 21.Kurosawa S, Yamaguchi T, Miyawaki S, et al. Prognostic factors and outcomes of adult patients with acute myeloid leukemia after first relapse. [Accessed May 23, 2013];Haematologica. 2010 95(11):1857–64. doi: 10.3324/haematol.2010.027516. Available at: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2966907&tool=pmcentrez&rendertype=abstract. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Hebart H, Brugger W, Grigoleit U, et al. Risk for cytomegalovirus disease in patients receiving polymerase chain reaction-based preemptive antiviral therapy after allogeneic stem cell transplantation depends on transplantation modality. [Accessed May 23, 2013];Blood. 2001 97(7):2183–5. doi: 10.1182/blood.v97.7.2183. Available at: http://www.ncbi.nlm.nih.gov/pubmed/11286223. [DOI] [PubMed] [Google Scholar]
- 23.Van der Heiden PLJ, Kalpoe JS, Barge RM, Willemze R, Kroes ACM, Schippers EF. Oral valganciclovir as pre-emptive therapy has similar efficacy on cytomegalovirus DNA load reduction as intravenous ganciclovir in allogeneic stem cell transplantation recipients. [Accessed May 23, 2013];Bone Marrow Transplant. 2006 37(7):693–8. doi: 10.1038/sj.bmt.1705311. Available at: http://www.ncbi.nlm.nih.gov/pubmed/16501590. [DOI] [PubMed] [Google Scholar]
- 24.Martinelli G, Trabetti E, Farabegoli P, et al. Early detection of bone marrow engraftment by amplification of hypervariable DNA regions. [Accessed May 24, 2013];Haematologica. 82(2):156–60. Available at: http://www.ncbi.nlm.nih.gov/pubmed/9175318. [PubMed] [Google Scholar]
- 25.Gray JPF, R J. A Proportional Hazards Model for the Subdistribution of a Competing Risk. 1999;94(446):496–509. [Google Scholar]
- 26.Barrett AJ. CMV: when bad viruses turn good. [Accessed May 25, 2013];Blood. 2011 118(5):1193–4. doi: 10.1182/blood-2011-06-354340. Available at: http://bloodjournal.hematologylibrary.org/content/118/5/1193.full. [DOI] [PubMed] [Google Scholar]
- 27.Behrendt CE, Rosenthal J, Bolotin E, Nakamura R, Zaia J, Forman SJ. Donor and recipient CMV serostatus and outcome of pediatric allogeneic HSCT for acute leukemia in the era of CMV-preemptive therapy. [Accessed May 26, 2013];Biol Blood Marrow Transplant. 2009 15(1):54–60. doi: 10.1016/j.bbmt.2008.10.023. Available at: http://www.bbmt.org/article/S1083-8791(08)00472-2/abstract. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Ito S, Pophali P, Co W, et al. CMV reactivation is associated with a lower incidence of relapse after allo-SCT for CML. [Accessed May 25, 2013];Bone Marrow Transplant. 2013 (November 2012):1–4. doi: 10.1038/bmt.2013.49. Available at: http://www.ncbi.nlm.nih.gov/pubmed/23562969. [DOI] [PMC free article] [PubMed]
- 29.Ruggeri L, Capanni M, Urbani E, et al. Effectiveness of donor natural killer cell alloreactivity in mismatched hematopoietic transplants. [Accessed May 26, 2013];Science. 2002 295(5562):2097–100. doi: 10.1126/science.1068440. Available at: http://www.sciencemag.org/content/295/5562/2097.abstract. [DOI] [PubMed] [Google Scholar]
- 30.Venstrom JM, Pittari G, Gooley Ta, et al. HLA-C-dependent prevention of leukemia relapse by donor activating KIR2DS1. [Accessed June 6, 2013];N Engl J Med. 2012 367(9):805–16. doi: 10.1056/NEJMoa1200503. Available at: http://www.ncbi.nlm.nih.gov/pubmed/22931314. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 31.Cooley S, Trachtenberg E, Bergemann TL, et al. Donors with group B KIR haplotypes improve relapse-free survival after unrelated hematopoietic cell transplantation for acute myelogenous leukemia. [Accessed June 7, 2013];Blood. 2009 113(3):726–32. doi: 10.1182/blood-2008-07-171926. Available at: http://bloodjournal.hematologylibrary.org/content/113/3/726.long. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 32.Foley B, Cooley S, Verneris MR, et al. Cytomegalovirus reactivation after allogeneic transplantation promotes a lasting increase in educated NKG2C+ natural killer cells with potent function. [Accessed May 25, 2013];Blood. 2012 119(11):2665–74. doi: 10.1182/blood-2011-10-386995. Available at: http://bloodjournal.hematologylibrary.org/content/119/11/2665.full. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 33.Fletcher JM, Prentice HG, Grundy JE. Natural killer cell lysis of cytomegalovirus (CMV)-infected cells correlates with virally induced changes in cell surface lymphocyte function-associated antigen-3 (LFA-3) expression and not with the CMV-induced down-regulation of cell surface class I HLA. [Accessed May 27, 2013];J Immunol. 1998 161(5):2365–74. Available at: http://www.ncbi.nlm.nih.gov/pubmed/9725232. [PubMed] [Google Scholar]
- 34.Thomson KJ, Mackinnon S, Peggs KS. CMV-specific cellular therapy for acute myeloid leukemia? [Accessed May 26, 2013];Blood. 2012 119(4):1088–90. doi: 10.1182/blood-2011-10-383943. author reply 1090–1. Available at: http://bloodjournal.hematologylibrary.org/content/119/4/1088.long. [DOI] [PubMed] [Google Scholar]
- 35.Junghanss C. Incidence and outcome of cytomegalovirus infections following nonmyeloablative compared with myeloablative allogeneic stem cell transplantation, a matched control study. [Accessed May 28, 2013];Blood. 2002 99(6):1978–1985. doi: 10.1182/blood.v99.6.1978. Available at: http://www.bloodjournal.org/cgi/doi/10.1182/blood.V99.6.1978. [DOI] [PubMed] [Google Scholar]
- 36.Maris M, Boeckh M, Storer B, et al. Immunologic recovery after hematopoietic cell transplantation with nonmyeloablative conditioning. Exp Hematol. 2003;31(10):941–52. doi: 10.1016/s0301-472x(03)00201-7. Available at: http://www.ncbi.nlm.nih.gov/pubmed/14550810. [DOI] [PubMed] [Google Scholar]