Risk factors for cytomegalovirus reactivation after CD6+ T-cell–depleted allogeneic bone marrow transplantation¹

Abstract

Background.  

Cytomegalovirus (CMV) infection is a significant cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT). Recipients of T-cell–depleted (TCD) transplants may be more susceptible to CMV infection as a result of the reduction in transferred T cell immunity. We sought to determine the effect of prior donor and patient CMV exposure on the incidence of CMV infection after TCD allogeneic HSCT.

Methods.  

We retrospectively examined CMV antigen testing results in all patients who had undergone CD6+ TCD related and unrelated donor allogeneic HSCT at our institution from 1996 to 1999. All 124 patients who had documented donor and recipient CMV serologies pretransplant and had undergone CMV antigen testing before day +100 posttransplant were included in the analysis.

Results.  

Forty-one percent of seropositive recipients and 1% of seronegative recipients developed evidence of CMV reactivation (odds ratio 54.1, 95% confidence interval [CI] 6.9–424.1, P <0.001). Prior donor CMV exposure did not place seronegative recipients at increased risk of CMV conversion. Multivariable analysis indicated that prior donor CMV exposure significantly reduced the risk of CMV reactivation in seropositive recipients by 81% (odds ratio 0.19, 95% CI 0.04–0.91, P =0.04). Grades II to IV acute graft-versus-host disease (GVHD) was associated with CMV conversion (P =0.04) when seropositive recipients underwent HSCT from CMV-negative donors, but not when the donor was CMV-seropositive (P =0.54).

Conclusions.  

The CMV serology status of the recipient, rather than the donor, was the primary determinant of risk for CMV conversion after TCD allogeneic HSCT. Despite CD6 + T-cell depletion, immunity against CMV seemed to be transferred with the donor graft and protected seropositive HSCT recipients from CMV reactivation.

Cytomegalovirus (CMV) infection remains a major cause of morbidity and mortality in patients undergoing allogeneic hematopoietic stem cell transplantation (HSCT) (^1–3). Ganciclovir (GCV) may help prevent the progression of CMV reactivation to CMV disease in allogeneic HSCT recipients, but GCV has significant bone marrow toxicity and can lead to increased risk of infection and treatment-related mortality (^4,5). Thus, much interest has focused on identifying allogeneic HSCT recipients who are at highest risk of developing CMV disease to formulate appropriate strategies for prophylaxis. Graft-versus-host disease (GVHD) and reduced T-cell immunity are factors that increase the risk of CMV disease (^3,6). Although T-cell depletion of the donor allograft reduces the likelihood of clinically significant GVHD, recipients of T-cell–depleted (TCD) allogeneic HSCT may be more vulnerable to CMV disease (^2,6). Although prior recipient exposure to CMV, as determined by recipient CMV seropositivity, has been shown to increase the risk of CMV disease, the effect of prior donor exposure to CMV is less clear (¹). In particular, the effect of donor CMV exposure on CMV-seropositive HSCT recipients has not been well studied. Although donor CMV seropositivity increases the risk of CMV disease in solid organ transplant recipients because of transmission of latent CMV in the transplanted organ, it is less clear whether a similar relationship exists in allogeneic HSCT (^7–10).

We retrospectively examined 124 patients who underwent TCD allogeneic HSCT at our institution to determine how the CMV serology states of donors and recipients influenced the risk of developing CMV antigenemia and disease. In particular, we were interested in examining how donor CMV serology status affected the probabilities of CMV reactivation and clinical disease in CMV-seropositive recipients.

MATERIALS AND METHODS

Patient Population

One hundred eighty-one consecutive patients underwent TCD allogeneic HSCT for hematologic malignancies at Brigham and Women’s Hospital (BWH) and Dana-Farber Cancer Institute (DFCI) on DFCI protocols 83044, 93003, 94055, 94110, 96022, 97217, and 97905 from January 1996 to December 1999. Information regarding patient and donor CMV serologies, donor source, number and timing of CMV antigen tests, CMV disease, relapse, GVHD, and demographic factors were obtained from patients’ charts and the institutional HSCT database. Patients were evaluable for analysis if they had documented pre-HSCT donor and recipient CMV serology tests and at least one CMV antigen test on or before day +100 posttransplant. One hundred twenty-four patients fulfilled these inclusion criteria.

Preparative Regimen and CMV Prophylaxis

The preparative regimen consisted predominantly of cyclophosphamide at 60 mg/kg infused on each of 2 consecutive days followed by 1400 cGy of total body irradiation (TBI) administered in fractionated doses of 200 cGy twice daily over 3.5 days. Newly harvested bone marrow, treated with anti-T12 (CD6) monoclonal antibody and rabbit complement as previously described, was the stem cell source in all transplants (¹¹). No other GVHD prophylaxis was given. To prevent transfusion-associated CMV transmission, all blood products were gamma-irradiated and filtered to remove leukocytes (^12,13). To reduce the number of donors to which HSCT recipients were exposed, all platelets for transfusion were obtained from single donors. All HSCT recipients were placed on prophylactic oral acyclovir; most patients received 400 mg t.i.d. (¹⁴). Patients who developed CMV reactivation were treated with intravenous GCV 5 mg/kg b.i.d. for 1 week, after which time the dose was reduced to 5 mg/kg q.d. for 3 more weeks. GCV dosing was adjusted as necessary for renal function. In addition, CMV-antigen converters underwent increased monitoring of their CMV-antigen states.

Determination of CMV-Antigen Status

CMV serology status of donors and recipients was determined before HSCT by ELISA measurement of CMV IgG. CMV reactivation was determined by the pp65 antigen assay, which was the prevalent test to detect CMV conversion at our institution from 1996 to 1999. Recipient CMV-antigen assays were performed by the virology laboratory at Children’s Hospital, as previously described (⁴). Briefly, CMV reactivation was determined by assay for the CMV matrix protein pp65 in peripheral blood leukocytes. Cytospin preparations of peripheral blood were incubated with pp65-specific monoclonal antibodies, and the number of positively staining cells per slide was measured. Slides were determined to be positive if the number of pp65 positive cells was above negative background. Slides were determined to be negative or positive, based on this assay, and no quantification of positive cells per slide was performed in this analysis.

Statistical Analysis

A logistic regression model was used to determine the probability of developing CMV disease while adjusting for the number of CMV tests on or before day +100 posttransplant. P values less than 0.05 were considered statistically significant, and P values less than 0.10 were deemed marginally significant. The likelihood ratio test was used to evaluate significance of the explanatory variables as predictors of CMV conversion.

RESULTS

Study Population

One hundred twenty-four patients met both inclusion criteria for our analysis and were evaluated (Table 1). For the group as a whole, the median age was 46 (range 20–65) years, and 43% of the patients were female. The most common diagnoses were acute myelogenous leukemia (AML) (37), acute lymphoblastic leukemia (ALL) (23), chronic myelogenous leukemia (CML) (20), non-Hodgkin’s lymphoma (NHL) (15), multiple myeloma (MM) (14), and myelodysplastic syndrome (MDS) (11). One hundred twenty (97%) of the donors were HLA-identical, and unrelated donors served as the marrow source in 44 of the transplants (35%). Thirty-seven percent of donors and 37% of recipients were CMV-seropositive before HSCT. Twenty-nine percent of the evaluable patients developed grades II to IV acute GVHD.

Table 1: Table 1. Characteristics of evaluable patients (n=124)

The reasons for exclusion of the remaining patients included unknown pretransplant CMV serology state of the donor or recipient (n=6) or absence of documented CMV-antigen testing before day +100 post-HSCT (n=51). A large number of these patients underwent transplantation during 1996, when CMV-antigen testing was new at our institution and was not commonly used. The characteristics of the 57 excluded recipients were examined and compared to those of the 124 evaluable subjects. The median age, patient gender, and distribution of underlying diagnoses were similar. Likewise, similar percentages of donors and recipients had been exposed to CMV, and the use of unrelated donors was not statistically different. The incidence of grades II to IV acute GVHD and overall survival rate were similar in the two groups.

CMV Reactivation and Disease

Twenty of the 124 evaluable patients (16%) developed CMV reactivation, as documented by at least one positive CMV-antigen test. Their characteristics are documented in Table 1. The median age and gender distribution were similar to those of the 104 patients who did not undergo CMV conversion. Fifty percent of the converters received marrow from unrelated donors compared with 32% of nonconverters (P =0.13). As described in Table 2, the median day of documented CMV reactivation was day +35 after transplant (range +19 to +129). Nine recipients (45%) converted between day 0 and +30, and 10 recipients (50%) converted between day +31 and +100. One recipient (5%) had CMV antigen reactivation after day +100. Six recipients eventually developed CMV disease; these six patients had a median day of conversion of day +30.5 (range +19 to +45). CMV disease was manifested as colitis (2), retinitis (2), pneumonia (1), and urinary disease (1). Nineteen patients (95%) were treated with intravenous GCV, and one patient was treated with oral acyclovir. Data on outcome of CMV therapy was available on 19 of 20 patients; therapy cleared CMV antigen in 16 patients (84%), whereas 3 patients (16%) had persistent CMV antigenemia despite appropriate treatment. Ten of 16 successfully treated patients remained CMV antigen-negative after therapy, but 6 of 16 had recurrent CMV antigenemia. Overall survival rate was 45% in converters and 54% in nonconverters (P =NS).

Table 2: Table 2. Characteristics of CMV converters (n=20)

The pre-HSCT donor and recipient serology results were examined for the 20 CMV-antigen converters (Table 2). Nineteen patients (95%) were CMV-seropositive entering transplant. Six of these 19 seropositive recipients received marrow from a seropositive donor, whereas 13 received stem cells from a CMV-negative donor. One CMV-antigen converter, who interestingly developed grade IV GVHD, was seronegative pre-HSCT and received marrow from a seronegative donor.

Effect of Donor and Recipient CMV Serologies on Incidence of CMV Conversion

As detailed in Table 3, 41% of the 46 seropositive recipients in our study developed CMV reactivation compared with only 1% of seronegative recipients, with an odds ratio (OR) of 54.1 (95% confidence interval [CI] 6.9–424.1, P <0.001). Donor CMV exposure did not increase the risk of CMV re-activation posttransplant. Thirteen percent of patients with CMV-seropositive donors experienced CMV reactivation posttransplant compared with 18% of patients with seronegative donors (P =0.48). Of the 25 seronegative recipients who underwent HSCT from a seropositive donor, none developed subsequent CMV-antigen conversion (Table 4). Thus, in our study no cases of CMV transmission via the donor allograft were observed in recipients who had no serological evidence of prior CMV exposure.

Table 3: Table 3. Prior recipient CMV exposure, but not donor exposure, is a risk factor for CMV-antigen conversion

Table 4: Table 4. Comparison of transplants by patient/donor CMV serology group

To account for multiple factors simultaneously, a logistic regression model was developed to adjust for age, gender, donor source (related or unrelated), donor CMV serostatus, and the number of antigen tests performed. Gender and age were insignificant (P =0.79), so a reduced logistic regression model adjusting for donor source, donor CMV serostatus, and number of antigen tests was used. Using this reduced multivariate analysis model, CMV-seropositive recipients were significantly less likely to develop CMV reactivation if a seropositive donor was used (OR 0.19, 95% CI 0.04–0.91, P =0.04).

The multivariate model showed that recipients of unrelated donor HSCT were more likely to develop CMV reactivation than patients who received stem cells from a related donor (OR 5.44, 95% CI 1.2–24.7, P =0.03). Donor CMV exposure was protective against CMV conversion in recipients of related (OR 0.10, 95% CI 0.01–0.95, P =0.05), but not unrelated (OR 0.63, P =0.99), donor bone marrow.

Effect of GVHD on CMV Conversion in Seropositive Recipients

Overall, patients who developed grades II to IV acute GVHD were not statistically more likely to develop CMV reactivation than patients with grade 0 or I acute GVDH (24% vs. 13%, P =0.18). However, seropositive recipients of seronegative donor marrow were more likely to undergo CMV reactivation if they developed grades II to IV acute GVHD (P =0.04); 5 of 5 patients with grades II to IV GVHD developed CMV conversion compared with 8 of 20 patients with grade 0 or I GVHD. The association between GVHD and CMV reactivation was not observed when a CMV-seropositive donor was utilized (P =0.54).

Effect of Number of CMV Tests

The number of CMV antigen tests per patient within the first 100 days of HSCT was examined to exclude a possible sampling bias. The median number of CMV tests was higher in the CMV converters (8, range 2–15) than in the nonconverters (4.5, range 1–14). However, the median number of negative tests before CMV reactivation in the 20 CMV converters was 1, and only 4 patients converted after more than 2 negative CMV tests. Seropositive recipients of seronegative donor HSCT underwent a similar number of CMV tests as did seronegative recipients of seropositive donor transplants (median 5 vs. 6), despite suffering a much higher incidence of CMV conversion (52% vs. 0%) (Table 4). Thus, the increased numbers of antigen tests in CMV converters were, in large part, because of increased testing after documented CMV reactivation.

DISCUSSION

Although donor seropositivity has long been recognized as a risk factor for CMV antigenemia and disease in recipients of solid organ transplants, the role of donor CMV exposure in allogeneic HSCT has been less clear. It was initially recognized in renal transplantation that latent CMV in cadaveric and living-related donor grafts can be transmitted to transplant recipients, resulting in clinical CMV disease in up to 80% of organ recipients (^7,9). Restriction enzyme analysis of viral DNA identified the donor grafts as the source of CMV reactivation (¹⁵). Similar findings were later reported with heart, lung, and liver transplants, and seronegative recipients of organs from seropositive donors seem to be at highest risk of severe CMV disease (^8,10). The risk of CMV transmission from a seropositive donor is considerably less, although not zero, in allogeneic HSCT. A recent study examined 115 consecutive HLA-identical sibling TCD HSCTs, including 12 seronegative recipients of stem cells from seropositive donors (¹). Two of these 12 patients (17%) developed CMV antigenemia, defined as 4 or more pp65-positive leukocytes per slide, which warranted GCV treatment. Twenty-eight of 68 seropositive recipients (41%) developed CMV reactivation, but the study did not provide information regarding any differences between the rates of CMV antigenemia in those seropositive recipients who received seronegative donor grafts and those who received seropositive donor marrow. Thus, although latent CMV may be transmitted from seropositive donor to seronegative recipient in allogeneic HSCT, the influence of CMV donor status upon the incidence of CMV reactivation in seropositive recipients remains unknown.

The results of our analysis suggest that immunity against CMV is transferred from seropositive donors to HSCT recipients and that this immunity prevents CMV reactivation in recipients who have had prior CMV exposure. Unlike solid organ transplants, in which latent CMV virus can be transmitted from donor to recipient, our study shows no increased risk of CMV conversion if a seropositive HSCT donor is used for a recipient with no prior CMV exposure. In fact, none of the 25 seronegative recipients of HSCT from previously exposed donors developed CMV conversion. Thus, our data indicate that prior CMV exposure should not exclude potential HSCT donors; in fact, CMV-seropositive donors are desirable if the transplant recipient has evidence of prior CMV exposure.

There is considerable evidence that T-cell immunity against CMV can be transferred with the donor graft in allogeneic HSCT. It is well established that donor lymphocyte infusion from CMV-seropositive donors can effectively treat CMV disease in recipients of TCD HSCT (^16,17). CD8+ T cells seem to play the primary role in mediating this immunity against CMV (^17,18), although effective treatment of CMV reactivation has also been reported using CD8-depleted donor lymphocyte infusion (¹⁹). It has been suggested that protection against CMV may be transferred despite T-cell depletion of donor stem cells. Although an older study of 40 CMV-seropositive recipients of HLA-matched, TCD allogeneic HSCT showed no significant difference in the incidence of CMV infections between recipients of seropositive and seronegative marrow (48% vs. 69%), the incidence of CMV pneumonia (15% vs. 61%) and CMV-related mortality (4% vs. 46%) were significantly lower in patients who received marrow from seropositive donors than in those who were transplanted from seronegative donors (²⁰). Thus, donor immunity to CMV seems to be critical in protecting CMV-seropositive allogeneic HSCT recipients against severe CMV infections.

Any analysis of studies of TCD allogeneic HSCT must take into account the different methods of T-cell depletion used in these studies. Single monoclonal antibody methods may achieve a 2 log₁₀ depletion of T cells, whereas multiple antibodies and physical methods, such as soybean lectin agglutination or counterflow centrifugal elutriation, may result in a 2.5 to 4 log₁₀ depletion (^11,21–25). As a result, TCD donor bone marrow usually contains between 10⁴ to 10⁶ T cells/kg. The extent of T-cell depletion has been shown to significantly influence the incidence rates of graft failure, GVHD, and disease relapse (^21,23,24). Various methods of T-cell depletion achieve removal of different numbers and types of T cells, as well as monocytes and natural killer (NK) cells that may also be important in GVHD and protective CMV immunity (²⁵). The degree of depletion of CD8+ lymphocytes may be particularly important, because CD8+ T cells seem to be the primary mediators of CMV immunity (^17,18). In a recent report, failure to recover CMV-specific CD8+ T cells identified seropositive TCD HSCT recipients who developed CMV antigenemia; in contrast, no recipient who recovered CMV-specific CD8+ lymphocytes experienced CMV reactivation (²⁶). Thus, the level of protection conferred by donor CMV immunity may depend on the degree of T-cell depletion and the specific populations of cells that are depleted with a particular method. A 2 log₁₀ depletion of CD6+ mature T cells in our study was able to reduce the incidence of GVHD while allowing transfer of CMV immunity from seropositive donors. The effect of other TCD methods on the risk of CMV disease remains unclear.

Prior donor CMV exposure protected against viral reactivation in seropositive recipients of related, but not unrelated, donor transplants. Two factors may account for this absence of protection in unrelated donor transplants. As mentioned previously, CMV immunity is likely transferred from donor to recipient via CMV-specific CD8+ T lymphocytes. However, recipients of unrelated donor TCD HSCT suffer delayed recovery and prolonged deficiencies of CD4+ and CD8+ T cells, compared with recipients of related donor TCD HSCT (²⁷). This delayed immune reconstitution in unrelated donor recipients may abrogate any protective antiviral effect seen in related-donor transplants. Secondly, CMV immunity may be strain-specific. Related donors and recipients are much more likely to have been exposed to identical CMV strains, and transferred donor CMV-specific CD8+ T cells should recognize recipient CMV with high affinity. However, transferred CMV-specific T cells from an unrelated donor are unlikely to encounter the same CMV strain in the recipient and may not recognize this different strain with adequate affinity.

The selection of patients who should receive CMV prophylaxis remains an area of active research. Recent advances in viral detection and therapy may change traditional recommendations regarding preemptive treatment for CMV. Viral load correlates with development of CMV disease in solid organ and bone marrow transplant recipients, and commercially available kits can measure CMV viral load by polymerase chain reaction or hybrid capture assay (^28–30). Although viral load generally correlates with pp65 antigen level (³⁰), some patients with high viral loads may have negative antigen tests; the clinical significance of this difference is unclear (²⁹). It is also unclear whether more sensitive detection methods will necessarily result in increased prophylactic therapy of CMV. The major drawback of current CMV drugs such as GCV, foscarnet, and cidofovir is toxicity, rather than lack of efficacy. However, new antiviral compounds with more favorable toxicity profiles may facilitate prophylactic treatment for CMV. The most promising new drug is valganciclovir, an orally administered prodrug of GCV with much greater bioavailability than oral GCV, a favorable safety profile, and equal efficacy to IV GCV in the treatment of CMV retinitis (³¹). The primary objective of CMV prophylaxis remains the prevention of clinical CMV disease with acceptable toxicity. Thus, identification of patients with minimal reactivation of CMV infection will be particularly valuable only when drugs with improved therapeutic windows can be developed.

In summary, the risk of CMV reactivation after TCD allogeneic HSCT is determined by the serology status of the recipient, and the use of a seropositive donor can decrease that risk. Despite blood product filtration and prophylactic antiviral therapy, 41% of CMV-seropositive recipients in our study developed CMV reactivation. Our analysis confirms that immunity against CMV can be transferred via the donor allograft in TCD HSCT. This transferred immunity protected previously exposed recipients of related-donor allogeneic HSCT from CMV reactivation. Thus, prior CMV exposure should not exclude potential donors for seronegative allogeneic HSCT recipients, and seropositive donors may be desirable for HSCT recipients who have previously been exposed to CMV. Further investigation should focus on the degree of protection that transferred CMV immunity provides for those seropositive HSCT recipients at highest risk of reactivation, particularly recipients of unrelated donor transplants.