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Blood, Vol. 95 No. 7 (April 1), 2000:
pp. 2240-2245
CLINICALOBSERVATIONS, INTERVENTIONS, AND THERAPEUTIC TRIALS
From the Department of Hematology, Department of Statistics,
Department of Immunology, and Department of Pathology, University
Hospital Rotterdam/Daniel den Hoed Cancer Center, Groene Hilledijk 301, 3075 EA Rotterdam, The Netherlands.
We evaluated the efficacy, toxicity, and outcome of preemptive
ganciclovir (GCV) therapy in 80 cytomegalovirus (CMV)-seropositive patients allografted between 1991 and 1996 and compared their outcome
to 35 seronegative patients allografted during the same period. Both
cohorts were comparable with respect to diagnosis and distribution of
high- versus standard-risk patients. All patients received a stem cell
graft from an HLA-identical sibling donor, and grafts were partially
depleted of T cells in 109 patients. Patients were monitored for CMV
antigenemia by leukocyte expression of the CMV-pp65 antigen. Fifty-two
periods of CMV reactivation occurring in 30 patients were treated
preemptively with GCV. A favorable response was observed in 48 of 50 periods, and only 2 patients developed CMV disease: 1 with esophagitis
and 1 with pneumonia. Ten of 30 treated patients developed GCV-related
neutropenia (less than 0.5 × 109/L),
which was associated with a high bilirubin at the start of GCV therapy.
Overall survival at 5 years was 64% in the CMV-seronegative cohort and
40% in the CMV-seropositive cohort (P = .01). Increased treatment-related mortality accounted for inferior survival. CMV seropositivity proved an independent risk factor for developing acute
graft-versus-host disease, and acute graft-versus-host disease predicted for higher treatment-related mortality and worse overall survival in a time-dependent analysis. We conclude that, although CMV
disease can effectively be prevented by preemptive GCV therapy, CMV
seropositivity remains a strong adverse risk factor for survival following partial T-cell-depleted allogeneic stem cell transplantation.
(Blood. 2000;95:2240-2245)
Cytomegalovirus (CMV) infection is an important cause
of morbidity and mortality in recipients of allogeneic stem cell
transplantation (SCT).1,2 CMV can cause primary infections
or may be reactivated in CMV-seropositive recipients and
CMV-seronegative recipients receiving a graft from a seropositive
donor.3 If untreated, CMV pneumonia may develop in up to
50% of bone marrow transplant (BMT) recipients showing virus
reactivation.1 The mortality of CMV pneumonia has remained
high despite the use of CMV-specific immunoglobulins and potent
antiviral agents such as ganciclovir (GCV).4-6 Therefore,
preventive measures are considered of utmost importance and have
received a great deal of interest. It has been shown that prophylaxis
with GCV effectively prevents CMV disease following allogeneic
transplantation.7-9 However, GCV prophylaxis administered
for a prolonged period after BMT has been complicated by severe
neutropenia, which itself is associated with an increased risk of
opportunistic infections and enhanced treatment-related mortality
(TRM).7-9 Alternatively, GCV may also be administered as
preemptive therapy based on the identification of CMV in blood or
bronchoalveolar lavage (BAL). Randomized and retrospective studies have
shown a significant reduction of CMV disease and enhanced survival in
CMV-seropositive donor-recipient pairs receiving GCV as preemptive
therapy while avoiding severe neutropenia.10-16 However, it
is still unclear how the outcome of HLA-matched sibling BMT in
CMV-seropositive patients receiving GCV preemptively compares to the
outcome of BMT in seronegative patients.
We set out to evaluate the efficacy, toxicity, and possible risk
factors of the preemptive use of GCV in CMV-seropositive patients,
initiated at first evidence of CMV antigenemia. Furthermore, survival,
TRM, and incidence of graft-versus-host disease (GVHD) in the
CMV-seropositive cohort were evaluated and compared to a cohort of
CMV-seronegative patients with a seronegative donor allografted during
the same period. The aim was to determine whether and how prior CMV
disease affects transplant outcome if GCV is used as preemptive therapy.
A total of 115 consecutive patients, who received an HLA-identical
sibling hemopoietic SCT at the Daniel den Hoed Cancer Center in
Rotterdam between 1991 and 1996, were included in the study. Two groups
of patients were defined: a group of 35 patients who were
CMV-seronegative before transplantation and received a graft from a
CMV-seronegative donor, hereafter designated as "CMV-seronegative patients," and a group of 80 patients in which either the
donor or the patient or both were CMV-seropositive (Table
1), hereafter designated as
"CMV-seropositive patients."
Transplantation
Supportive care
Diagnostic tests for CMV reactivation and CMV disease The presence of CMV lower matrix protein pp65-positive leukocytes in peripheral blood or BAL fluid was analyzed as described.17,18 In short, cytospin preparations were prepared and incubated with pp65-specific monoclonal antibodies: 1 slide with clone 1C3/A-YM-1 (Biogenesis, Bournemouth, UK) and another slide with clone C10/C11 (Biotest, Seralco, Brussels, Belgium). In addition, 1 cytospin slide was used as a negative control, and slides with CMV-infected granulocytes served as positive controls. Staining was performed using the alkaline phosphatase-antialkaline phosphatase method (dilution 1:50; Serotec, Oxford, UK). CMV disease was diagnosed on the basis of an inflammatory process due to CMV, confirmed either by the immediate early antigen (IEA) assay or CMV cultures, and preferably combined with the presence of typical cytopathic effects in histologic preparations if biopsies were available. All biopsies or leukocytes obtained by BAL were cultured for CMV for 14 days. Histologic examination of tissue biopsies included immunohistochemical analysis for CMV using pp65-specific monoclonal antibody clone 1C3 (Biogenesis). Pneumonia was classified as idiopathic when (multiple) biopsies of lung tissue showed interstitial inflammation but without any positive indication of viral, bacterial, parasitic, or fungal causes upon specific culture and immunohistochemical analysis.Ganciclovir therapy CMV-seropositive patients were monitored weekly from transplantation until day 150 for CMV antigenemia. Test results were considered positive in case of at least 1 positively staining leukocyte. Patients with a positive antigenemia test were monitored twice weekly. Preemptive GCV therapy was initiated (5 mg/kg of body weight intravenously, twice daily) if 4 or more positive leukocytes were identified by the IEA assay. Treatment was discontinued after 2 successive negative test results, which was considered a favorable response, if no CMV disease had developed during that time. That specific protocol of preemptive therapy was chosen to avoid GCV treatment in patients with low-grade antigenemia, who may resolve their antigenemia spontaneously, and to avoid GCV treatment in false-positive antigenemia and thereby prevent GCV-associated neutropenia while preserving effective prevention of CMV disease.Statistical analysis Patients were analyzed for response of CMV reactivation to GCV treatment. Patient characteristics in the 2 cohorts were compared using Pearson's 2 test or the Wilcoxon rank sum test,
whichever was appropriate. All reported P values are 2-sided,
and a significance level of  = .05 was used. Overall survival was
measured from transplantation until death from any cause. Patients
still alive at the time of analysis were censored at the last follow-up
date. TRM was determined from the date of transplantation until death
related to the transplantation. Patients who died from other causes
were censored at the date of death. Time to acute GVHD grades 2-4 was
calculated from date of transplantation until occurrence of acute GVHD.
Patients who died before day 100 posttransplant without having suffered
from acute GVHD were censored at the date of death. Patients without GVHD and still alive at day 100 were then censored. Time to acute GVHD
grades 2-4, overall survival, and TRM were estimated by the Kaplan-Meier method. The following variables were included in the
analysis of prognostic factors: sex, age, risk status, CMV serostatus
of the patient/donor (both negative vs at least 1 positive), and graft
characteristics (number of nucleated cells, CFU-GM
[granulocyte-macrophage colony-forming units] and CD3+ T
cells infused). Univariate survival analysis was performed using the
log-rank test to see whether there was a difference in survival between
the subgroups, and univariate Cox regression was used to determine
whether the relation was monotonous. The variables that appeared
significant in the univariate Cox regression were also used in a
multivariate Cox regression. Moreover, a Cox regression analysis, with
occurrence of acute GVHD grades 2-4 included as a time-dependent
covariate, was performed to examine whether acute GVHD predicted for
higher TRM and worse overall survival.
A total of 115 consecutive HLA-identical sibling BMTs were evaluated. Patient characteristics are presented in Table 1. Two cohorts of patients are presented: a CMV-seronegative cohort and a seropositive (donor, recipient, or both) cohort. Thirty-five patients and their donors were CMV-seronegative. Eighty recipient-donor pairs were CMV-seropositive (the so-called "CMV-seropositive patients"), including 68 seropositive recipients and 51 seropositive donors. In 39 cases, both donor and recipient were seropositive. High-risk patients were distributed equally among the CMV-seronegative and CMV-seropositive cohorts, and diagnoses did also not differ between the 2 cohorts. The median age of the CMV-seropositive cohort was 43 years and exceeded that of the seronegative cohort, which was 37 years (P = .02). Median numbers of mononuclear cells, CFU-GM, and T cells infused did not differ between the CMV-seronegative and CMV-seropositive cohort. Partial TCD of the graft was applied in 109 patients, and 6 patients received an unmanipulated graft because these patients suffered from high-risk disease and were considered to be at high risk for relapse. Alternatively, they received cyclosporine and methotrexate for prevention of GVHD. Ganciclovir treatment A total of 47 patients developed CMV antigenemia as defined by at least 1 pp65-positive peripheral blood leukocyte. Thirty of these patients showed an increase in the number of positive leukocytes up to 4 or more positive leukocytes, which was the threshold for initiating GCV treatment. Fifty-two periods of CMV antigenemia occurring in these 30 patients were treated with GCV. Two of these 30 patients were CMV-seronegative before transplantation but received a graft from a seropositive donor. Seventeen patients developed so-called low-grade antigenemia their IEA test results showed less than 4 positive leukocytesand, by protocol, these patients were
not treated with GCV. None of them developed CMV disease.
CMV-seropositive patients with versus without CMV antigenemia did not
differ with respect to their graft characteristics (mononuclear cells,
CFU-GM, T cells), age, sex, underlying disease, or risk status (results
not shown). Results of the 30 patients treated with GCV are presented
in Table 2. Two patients died before the effects of GCV could be evaluated. The median duration of GCV treatment
was 10 days (range, 2-38 days). A favorable response (2 consecutive
negative antigenemia test results and no signs of CMV disease) was
observed in 48 of 50 (96%) evaluable treatment courses. One patient
developed a CMV pneumonia that was lethal, and another patient
developed a CMV esophagitis necessitating the addition of CMV-specific
immunoglobulins combined with a prolonged course of GCV therapy.
Recurrence of CMV antigenemia was observed 22 times in 12 patients.
These recurring episodes of antigenemia all responded favorably to a
second, third, fourth, or fifth course of GCV. In addition
to the 2 aforementioned patients with CMV pneumonia and esophagitis, 1 other patient developed a CMV pneumonia, which was not preceded by
peripheral blood CMV antigenemia. No CMV disease was observed after day
100.
Survival, TRM, and GVHD Overall survival of these 115 patients was 64% ± 4% at 1 year and 47% ± 5% at 5 years posttransplant. The median follow-up for patients still alive was 43 months (range, 9-95 months). Survival was not significantly affected by diagnosis, risk status, sex, and graft characteristics (such as numbers of CFU-GM, T cells, and mononuclear cells infused). In univariate analysis, both positive CMV serology (of donor or recipient) and older age predicted for inferior survival. However, only CMV serology significantly affected survival (P = .03) in multivariate Cox regression analysis. The seropositive cohort included also 12 "-/+" patientsthose who were seronegative before SCT but
received a graft from a positive donor. Overall survival was
45% ± 9% in these 12 patients at 36 months after SCT, which did
not differ from 39 +/+ patients and 29 +/- patients, who completed the
CMV-seropositive cohort (Table 1). In contrast, overall survival was
70% ± 8% in the seronegative group at 36 months after SCT
(Figure 1).
In this study, we show that CMV seropositivity continues to
represent a major adverse risk factor for transplant-related morbidity and mortality despite the very efficient prevention of CMV disease by
preemptive GCV therapy in patients receiving a partial TCD graft.
Increased TRM accounted for inferior survival observed in
CMV-seropositive patients versus CMV-seronegative patients. Furthermore, an increased incidence of acute GVHD was observed in
CMV-seropositive patients, and the development of acute GVHD strongly
predicted for TRM and overall survival. Thus, CMV disease prior to
transplantation does not affect transplantation outcome by CMV disease
itself but, rather, by increasing the incidence of acute GVHD.
Submitted May 21, 1999; accepted December 1, 1999.
Reprints: J.J. Cornelissen, Daniel den Hoed Cancer Center,
Department of Hematology, Groene Hilledijk 301, 3075 EA Rotterdam, The Netherlands.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
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This article has been cited by other articles:
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Top
Abstract
Introduction
3 days until 100 days after BMT. Cyclosporine was combined with methotrexate (15 mg/m2 on day 1; 10 mg/m2 on days 3, 6, and 11)
in case of an unmanipulated graft. The source of hematopoietic stem
cells was bone marrow in 105 patients and peripheral blood stem cells
in 10 patients. The institutional review board approved the protocols,
and patients provided informed consent. Graft characteristics are
presented in Table 
