Biology of Blood and Marrow Transplantation
Volume 12, Issue 5 , Pages 573-584, May 2006

Reduced Incidence of Acute and Chronic Graft-versus-Host Disease with the Addition of Thymoglobulin to a Targeted Busulfan/Cyclophosphamide Regimen

Fred Hutchinson Cancer Research Center and University of Washington School of Medicine, Seattle, Washington

Received 23 November 2005; accepted 17 December 2005.

Article Outline

Abstract 

To reduce the incidence of graft-versus-host disease (GVHD), we added Thymoglobulin (THY) to dose-adjusted oral busulfan plus cyclophosphamide (targeted BUCY). The starting dose of THY was 4.5 mg/kg given over days −3, −2, and −1, escalated in steps of 1.5 mg/kg in cohorts of 15 evaluable patients. Escalation was dependent on acute GVHD incidence and Epstein-Barr virus reactivation. Fifty-six patients with myelodysplastic syndrome and other myeloid disorders underwent transplantation with peripheral blood progenitor cells from related (n = 30) or unrelated (n = 26) donors. All but 2 patients achieved engraftment, and 56% survived in remission beyond 1 year. The incidence of acute GVHD was 50%, and that of chronic GVHD was 34%. The highest THY dose was 6.0 mg/kg, a dose at which 1 patient experienced Epstein-Barr virus reactivation. Nine patients did not receive the prescribed THY dose. Results were comparable for related and unrelated transplants and for patients given 4.5 or 6.0 mg/kg THY. Among 27 myelodysplastic syndrome patients (14 with related and 13 with unrelated donors) who underwent transplantation concurrently with targeted BUCY without THY, the incidence of acute and chronic GVHD was 82%. Thus, THY 4.5 to 6.0 mg/kg seemed beneficial for GVHD prevention in BUCY-conditioned patients who underwent transplantation with peripheral blood progenitor cells, although relapse-free survival did not differ significantly from that in comparable historical controls not given THY.

Key words:  Thymoglobulin , Graft-versus-host disease , Peripheral blood progenitor cells , Busulfan

 

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Introduction 

Allogeneic hematopoietic cell transplantation (HCT) is currently the only therapy with curative potential for myelodysplastic syndrome (MDS) and myeloproliferative disorders. Depending on the disease state at the time of transplantation, 40% to 70% of patients survive long-term in remission and are probably cured of their disease [1, 2, 3]. The results with transplants from unrelated donors selected for HLA identity on the basis of high-resolution typing are approaching those achieved with HLA genotypically identical sibling transplants, thus making this treatment available to more patients [1]. However, graft-versus-host disease (GVHD) has remained a major problem in patients both with related and unrelated donors, affecting as many as 70% to 80% of patients and causing considerable morbidity and mortality, often related to infections. In addition, among patients with more advanced disease, posttransplantation relapse is a frequent complication [1, 3, 4, 5]. In several studies, intensification of the conditioning regimens has reduced relapse rates, but generally at the expense of a higher incidence of regimen-related toxicity and mortality [6, 7]. Furthermore, the cumulative incidence of GVHD tends to be higher with more intensive conditioning regimens [8, 9], and intensification of GVHD prophylaxis—for example, by in vitro T-cell depletion of the transplant inoculum—has been associated with an increase in the posttransplantation relapse rate [10].

Several transplant teams [11, 12, 13, 14] incorporated the rabbit antihuman thymocyte globulin Thymoglobulin (THY) Genzyme, Cambridge, MA into conditioning regimens and observed a lower incidence of GVHD than with otherwise comparable regimens without THY. Some reports also suggested that even if GVHD developed, treatment was more successful [14]. We therefore conducted a prospective trial in patients with high-risk myeloid disorders, using a well-established conditioning regimen (oral busulfan [BU] targeted to steady-state plasma levels of 800-900 ng/mL given over 4 days, followed by cyclophosphamide [CY] 60 mg/kg on 2 consecutive days [1]) to which THY was added on days −3, −2, and −1 before transplantation in a dose-escalation design. The purpose was to determine the optimum dose of THY for GVHD prevention without increasing the risk of relapse and infections. In a parallel study, patients with low-risk MDS were conditioned with an identical BUCY regimen but did not receive THY. We report here the results of both trials and compare them with those in a historical control group.

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Patients and methods 

Patients 

Patient and disease characteristics are summarized in Table 1.

Table 1. Patient and Disease Characteristics
VariableRegimen
tBUCY/THYtBUCY (Concurrent Control)tBUCY (Historical Controls) [1]
No. patients studied562793
Patient age, y, range (median)9-65 (50)11-64 (51)7-66 (47)
Sex (M/F)24/3217/1045/48
Diagnosis
RA4⁎26†55
RARS—1†2
RAEB14‡—20
AML17§—10
MDS (NOS)3∥——
ICMF8——
Other MPD6#—4
CMML4⁎⁎††—2
Interval from diagnosis to transplantation, mo, range (median)1.5-318 (6.1)3.5-146 (11.8)1-128 (10)
Comorbidity score (HCT-CI), range (median)0-7 (1)0-4 (1)NA

AML indicates acute myelogenous leukemia; CMML, chronic myelomonocytic leukemia; ICMF, idiopathic chronic myelofibrosis (agnogenic myeloid metaplasia); MDS, myelodysplastic syndrome; MPD, myeloproliferative disorder; NOS, not otherwise specified; RA, refractory anemia; RAEB, RA with excess blasts; RARS, RA with ringed sideroblasts; tBUCY, targeted busulfan + cyclophosphamide; THY, Thymoglobulin; HCT-CI, hemopoietic cell transplantation comorbidity index [33]; NA, not available (HCT-CI developed only since then).

⁎ All had intermediate- or high-risk cytogenetics by International Prognostic Scoring System (IPSS) criteria; 3 were intermediate 1, and 1 was intermediate 2 risk; 1 patient had refractory cytopenia with multilineage dysplasia (RCMD) by World Health Organization (WHO) criteria.

† Six patients were classified as low risk and 21 as intermediate 1 risk by IPSS; 19 patients had good-risk (14 normal) and 8 intermediate-risk karyotypes. By WHO criteria, 8 patients had RA, 12 had RCMD, 6 patients had MDS-U undefined as per WHO criteria, and 1 patient had RARS.

‡ By IPSS classification, 1 patient was categorized as low risk, and 4 were classified as intermediate 1, 6 as intermediate 2, and 3 as high risk. Among these, 2 had good-risk (all normal), 5 intermediate-risk, and 7 high-risk karyotypes; 8 had RAEB1 and 6 RAEB2 by WHO criteria.

§ Ten of these had transformed to AML from MDS, and 7 had de novo AML (4 in first complete remission, 1 in second complete remission, and 2 with refractory disease). Among the patients with de novo AML, 6 had normal karyotypes, and 1 had a complex karyotype.

∥ By IPSS, 2 patients were classified as intermediate 1 and 1 was classified as high risk; 2 had normal and 1 complex cytogenetics.

All 14 patients with ICMF or other MPD had severe fibrosis [39]; by the criteria of the Lille classification, 3 patients qualified as good risk and 11 as intermediate risk [40].

# Three patients had a history of polycythemia vera and 2 of essential thrombocythemia; 1 patient showed >5% marrow myeloblasts and could not be classified.

⁎⁎ One patient was simultaneously diagnosed with chronic lymphocytic leukemia.

†† Using the criteria of the M.D. Anderson classification, 2 patients had low-risk and 1 each had intermediate 2 and high-risk CMML (karyotypes were normal, +8, 20q−, and complex, respectively).

Targeted BUCY + THY (Protocol 1) 

From November 2002 through August 2004, 56 patients (24 male and 32 female), 9 to 63 years (median, 50 years) of age, were enrolled. The interval from diagnosis to transplantation was 1.5 to 318 months (median, 6.1 months). There were 31 patients with a diagnosis of MDS (see Table 1 for categories), including 10 who had transformed to acute myeloid leukemia (AML); 14 patients with myeloproliferative disorders; 7 patients with de novo AML; and 4 patients with chronic myelomonocytic leukemia.

Targeted BUCY (Protocol 2; Referred to as Concurrent Control) 

From December 2001 through May 2004, 27 patients (17 male and 10 female), 11 to 64 years (median, 51 years) of age, with low-risk MDS were enrolled (see Table 1 for categories). The interval from diagnosis to transplantation was 3.5 to 146 months (median, 11.8 months). None of the patients had high-risk cytogenetic findings, as defined by the International Prognostic Scoring System [15].

Historical Controls 

In addition, we used for comparison data from 93 historical control patients conditioned with targeted BUCY (tBUCY) who were reported previously [1]. These patients were 7 to 66 years (median, 47 years) old, 45 were male, and 48 were female. The interval from diagnosis to transplantation was 1 to 128 months (median, 10 months). The diagnoses are listed in Table 1. Comorbidity indices were not available on those patients.

Conditioning Regimens 

The conditioning regimens are summarized in Table 2.

Table 2. Donor and Transplant Characteristics
VariableRegimen
tBUCY/THYtBUCY
Donor age, y, range (median)8-63 (42)27-69 (40)
Donor sex (M/F)29/2712/15
Donor/patient relationship
HLA-identical sibling3014
Unrelated volunteer26⁎13†
Source of stem cells
Bone marrow1‡0
Peripheral blood5527
Donor/patient CMV serology
+/+1610
+/−83
−/+165
−/−169
Conditioning
Targeted BUCY5627
Thymoglobulin
4.5 mg/kg150
6.0 mg/kg32§0
Incomplete dose (0.5-3.5 mg/kg)90
GVHD prophylaxis MTX/CSP5627

CMV indicates cytomegalovirus; CSP, cyclosporine; MTX, methotrexate; tBUCY, targeted busulfan + cyclophosphamide; THY, Thymoglobulin.

⁎ Among these, 3 were 1 allele–mismatched with the patient (see text).

† Among these, 3 were 1 antigen– or 1 allele–mismatched with the patient (see text).

‡ Donor refused G-CSF administration.

§ In the first cohort of 15 patients who received the prescribed dose of 6 mg/kg, 1 patient showed a titer increase of Epstein-Barr virus DNA copies in plasma >1000; per protocol, this prevented a further dose escalation of THY, and subsequent patients were treated at doses of 6 mg/kg. No further Epstein-Barr virus activation was observed.

Targeted BUCY + THY 

Patients were prepared with a regimen of oral BU at a starting dosage of 1 mg/kg to be given every 6 hours for 16 doses on days −7 to −4, followed by CY intravenously 60 mg/kg/d on days −3 and −2. Phenytoin was given as prophylaxis for potential BU-induced seizures as described previously [16]. Details of the chemical and pharmacokinetic analysis of BU to provide accurate assessment of BU exposure during conditioning have been described previously [17]. Blood samples were collected at 0, 1, 2, 4, and 6 hours after the morning doses on days 1, 2, and 3, and subsequent doses were adjusted to achieve steady-state plasma levels in the range of 800 to 900 ng/mL, as described previously [16]. No dose adjustments for CY were made. On days −3, −2, and −1, patients received THY intravenously. The starting dose of THY was 4.5 mg/kg total (0.5 mg/kg on day −3 and 2 mg/kg each on days −2 and −1). Plasma samples were obtained before the first dose of THY, 1 hour after completion of the third dose, and in the morning after the completion of hematopoietic stem cell infusion (day +1).

Patients were enrolled in cohorts of 15, with THY starting at a dose of 4.5 mg/kg. If <5 patients developed acute GVHD grades II to IV and no patient showed reactivation of Epstein-Barr virus (EBV), as determined by an increase in DNA copies in blood plasma to >1000/mL, then the next cohort was to receive the same dose of THY. If ≥5 patients developed acute GVHD, then the dose was to be increased to 6 mg/kg. The next cohort of 15 was to receive a dose of 7.5 mg/kg or the same dose (6 mg/kg), depending on the frequencies of GVHD and EBV reactivation, a complication reported in one earlier study [18].

Targeted BUCY 

Patients received the same regimen of tBUCY as described previously, but without the addition of THY.

Donors and Source of Stem Cells 

Donor characteristics are summarized in Table 2. Among 56 patients prepared with tBUCY plus THY, 30 received transplants from genotypically HLA-identical siblings, and 26 from unrelated donors. All sibling pairs were identical for HLA-A, -B, -C, and -DQB1 by intermediate-resolution typing and for -DRB1 by sequencing. For unrelated transplants, all patients and donors had DNA sequenced for HLA-A, -B, -C, and -DRB1 and had intermediate-resolution typing for -DQB1, except for 4 donors who had sequencing at -A, -B, and -DRB1 but intermediate-resolution typing for -C and -DQB1. Three donors were mismatched with the patient for a single allele (-B, -A, or -DQB1); all others were HLA identical. All patients but 1 received granulocyte colony-stimulating factor (G-CSF)–mobilized peripheral blood progenitor cells (PBPCs).

Among 27 concurrent controls prepared with tBUCY and no THY, 14 received transplants from genotypically HLA-identical siblings, and 13 from unrelated donors (all had DNA sequenced at HLA-A, -B, -C, and -DRB1 with intermediate-resolution typing for -DQB1). Three were mismatched with the patient (1 for a -DRB1 antigen and 2 for a -B allele), and 10 were HLA identical to the patient. All patients were given G-CSF–mobilized PBPCs.

GVHD Prophylaxis and Therapy 

Prophylaxis for GVHD, in addition to the pretransplantation administration of THY, consisted of methotrexate given at 10 mg/m2 on days 1, 3, 6, and 11 and cyclosporine (CSP) starting on day −1 and, in the absence of GVHD, continued on a daily basis until day 180 as described previously [19]. If GVHD was present, CSP was continued beyond day 180 for as long as necessary. The diagnoses of acute and chronic GVHD were made by established criteria [20]. Methylprednisolone (or prednisolone) was given as first-line therapy. Steroid-refractory GVHD was treated according to protocols active at the time of the patients’ transplantations.

Engraftment and Rejection 

The day of engraftment was defined as the first of 3 consecutive days on which the neutrophil count exceeded 0.5 × 109/L [21]. Evidence of graft rejection was sought when the absolute neutrophil count failed to reach 0.5 × 109/L, when the absolute neutrophil count declined after initial recovery, and when relapse was diagnosed. Patients who died before day 28 were not evaluated for engraftment. When the donor and patient were of different sexes, engraftment was verified by testing bone marrow and peripheral blood mononuclear cells with fluorescence in situ hybridization with Y or X chromosome–specific probes [22, 23]. When patient and donor were of the same sex, DNA from marrow and peripheral blood mononuclear cells was amplified for several variable number tandem repeat loci for identification of informative host and donor markers [24].

Relapse 

All patients had marrow samples examined morphologically and by cytogenetic and flow cytometric analyses on days 28 and 84 after transplantation and then annually or as clinically indicated. Relapse was defined morphologically by marrow dysplasia and cytogenetically by the detection of metaphases in the marrow that showed the same clonal marker(s) as identified before transplantation (or by fluorescence in situ hybridization markers, if appropriate) or by the reemergence of myeloblasts or aberrant precursors identified by flow cytometry [25].

Infections 

Blood samples were examined weekly for evidence of cytomegalovirus (CMV) antigenemia [26]. Interstitial pneumonia was diagnosed on the basis of radiographic findings; by use of culture, histologic, or histochemical analysis of bronchoalveolar lavage fluid; by open lung biopsy; or at autopsy [27]. Strategies to prevent infectious diseases included the prophylactic or preemptive use of systemic antibiotics, fluconazole, acyclovir, and ganciclovir (or foscarnet) [28]. All CMV-seronegative patients received CMV-negative or leukocyte-depleted blood products. Acyclovir prophylaxis was given to all patients who were seropositive for herpes simplex or herpes zoster virus. Plasma samples were also monitored twice weekly (through day 42) for EBV DNA in plasma, and treatment with rituximab was given if the titer increased to 1000 copies per milliliter [29].

Causes of Death 

Deaths that occurred after progression or recurrence of the underlying disease were categorized as due to disease recurrence, regardless of the proximate causes; deaths in the absence of relapse were categorized as nonrelapse mortality. Infection was considered the cause of death when a bacterial, viral, or fungal infection other than interstitial pneumonia was the proximate cause of death in patients who had not relapsed. Infections were further categorized according to whether or not they were associated with GVHD and with organ failure. Multiorgan failure was considered the cause of death if decompensation occurred in at least 2 organ systems (eg, liver and kidneys or liver and lungs) and could not be attributed to GVHD or infection alone.

Statistical Methods 

The probabilities of survival and relapse-free survival were estimated by using the Kaplan-Meier method. The incidence rates of relapse, acute and chronic GVHD, nonrelapse mortality, and infection were expressed in terms of cumulative incidence [30]. Cox regression was used to analyze risk factors related to the hazards for these outcomes. In these analyses, mortality was considered a competing risk for all other outcomes, and relapse was a competing risk for nonrelapse mortality. The effect of THY doses on serum levels of THY was analyzed by the Wilcoxon rank sum test.

To put these results into perspective, outcomes were also compared with previously reported results in patients with MDS or myeloproliferative disease in various risk categories conditioned with tBUCY without the use of THY (historical controls) [1]. Data were analyzed as of August 31, 2004.

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Results 

Conditioning 

The target range for steady-state plasma levels of BU was 800 to 900 ng/mL. The actual BU steady-state concentration levels reached in patients conditioned with tBUCY plus THY were 734 to 1081 ng/mL (median, 874 ng/mL). The median total dose of BU administered was 14.9 mg/kg (range, 9.8-22.4 mg/kg). Among the 27 concurrent patients prepared with tBUCY, BU steady-state concentration levels were 776 to 1074 ng/mL (median, 921 ng/mL). The median total dose of BU administered was 14.2 mg/kg (range, 11.2-19.8 mg/kg).

The starting dose of THY was 4.5 mg/kg (over 3 days) for a cohort of 15 patients. Two patients did not receive the prescribed dose (because of severe chills and patient preference). Thus, 17 patients were enrolled in this cohort. None showed EBV reactivation, but 7 of 15 evaluable patients required treatment for acute GVHD, and the THY dose was increased to 6 mg/kg. To arrive at 15 evaluable patients in this cohort, 20 patients had to be enrolled, because 5 did not receive the prescribed dose of THY (because of hypotension or patient decision in 1 patient each and attending physician preference in 3). One of 15 patients given the complete dose of THY experienced EBV reactivation. Thus, there was no further dose escalation of THY, and 19 additional patients were enrolled, 17 of whom received THY at 6 mg/kg, whereas in 2, the dose on day −1 was not given (because of hypotension and patient preference, respectively).

In 42 patients who received the prescribed dose of THY, we also determined serum levels of THY by using an assay developed by Genzyme (Cambridge, MA). Levels in samples obtained upon completion of the last dose of THY (day −1) were 3.9 to 80.1 μg/mL (median, 29.3 μg/mL) for total THY and 0.2 to 7.2 μg/mL (median, 2.3 μg/mL) for “active” THY, defined as the amount that was available for binding to lymphocytes, as determined by flow cytometry [31]. The corresponding levels after completion of PBPC infusions (day +1) were 10.2 to 68.8 μg/mL (median, 29.8 μg/mL) and 0.5 to 4.2 μg/mL (median, 1.9 μg/mL), respectively. In patients given 4.5 mg/kg THY, the median level on day −1 was 24.7 μg/mL for total and 2.0 μg/mL for active THY; the corresponding levels in patients given 6.0 mg/kg were 40.9 and 2.8 μg/mL, respectively. The differences at each dose level were significant (P = .02 and .03, respectively). Levels obtained after the completion of PBPC infusion (day +1) also tended to be higher among patients who received the higher dose of THY, but differences were not significant (not shown).

Engraftment 

Among tBUCY + THY–treated patients, 2 died (on days 19 and 22, respectively) and could not be evaluated for engraftment. Among the remaining 54 patients, 1 with an HLA-identical sibling donor, and 1 with an unrelated donor did not have sustained engraftment. All concurrent control patients prepared with tBUCY survived beyond day 28 and achieved sustained engraftment.

Graft-versus-Host Disease 

Results are illustrated in Figure 1. Among patients treated with tBUCY + THY, 52 (24 with unrelated and 28 with HLA-identical sibling donors) were evaluated for acute GVHD (Figure 1A). Overall, 30 patients (16 unrelated and 14 related transplant recipients) developed acute GVHD (grades II-IV) necessitating therapy, for a cumulative incidence of 50% (by day 100). Among the 44 patients who received the prescribed dose of THY, 9 of 22 with related and 14 of 22 with unrelated donors required therapy for acute GVHD, for an overall incidence of 44%. Among 8 evaluable patients who did not receive the prescribed dose of THY (1 of these did not achieve engraftment), 7 (88%) required therapy for GVHD. The cumulative incidence of chronic GVHD at 1 year was 34% (31% among patients who had received the prescribed dose of THY). The incidence of chronic GVHD by THY dose is shown in Figure 1B. There was no significant difference in the incidence of acute or chronic GVHD between patients treated with 4.5 or 6.0 mg/kg THY. There was also no difference with regard to the severity of GVHD between the 2 groups.

  • View full-size image.
  • Figure 1. 

    Cumulative incidence of GVHD: acute (A) and chronic (B). Shown separately are the incidences for patients given the prescribed doses of 4.5 or 6.0 mg/kg THY and patients who did not receive the prescribed dose (THY <4.5 mg/kg). The overall incidence of acute GVHD (grades II-IV) was 50%, and the overall incidence of extensive chronic GVHD was 34%.

Among concurrent patients conditioned with tBUCY without THY, 11 (79%) of 14 with sibling donors and 11 (85%) of 13 with unrelated donors developed acute GVHD necessitating therapy. Extensive chronic GVHD requiring therapy developed in 82% of patients. One patient in this study had neither acute nor chronic GVHD.

Survival 

One-year relapse-free survival was 56% among the 56 patients with more advanced myeloid malignancies who were conditioned with tBUCY + THY and was 78% in concurrent controls (all patients with good-risk MDS) conditioned with tBUCY only. Among patients who survived beyond 1 year, there were no significant differences in the Karnofsky performance scores between tBUCY/THY-conditioned and tBUCY-conditioned concurrent controls, although only 2 (6%) of 31 tBUCY/THY-conditioned patients had scores of <80, compared with 5 (24%) of 21 patients not given THY.

Relapse 

The tBUCY + THY protocol enrolled patients with advanced or high-risk MDS, myeloproliferative disorders, and AML. Twelve of the 56 patients had disease recurrence, for a cumulative incidence of 21%. The corresponding incidence rates for patients who received 4.5 mg/kg versus 6.0 mg/kg versus <4.5 mg/kg (incomplete dose) THY were 20%, 13%, and 44%, respectively. Among patients with refractory anemia with excess blasts, the cumulative incidence of relapse at 1 year was 35%, compared with 24% for patients with AML and 14% for patients with myeloproliferative disorders. There was a trend for BU levels <800 ng/mL to be associated with a higher relapse rate than with levels >800 ng/mL (hazard ratio, 3.5; P = .17). All concurrent controls conditioned with tBUCY without the addition of THY had good-risk MDS (<5% marrow myeloblasts and no high-risk cytogenetics), and none experienced disease recurrence.

Nonrelapse Mortality 

Causes of death are listed in Table 3. Among patients conditioned with tBUCY + THY, nonrelapse mortality was 12.5% at day 100 and 23% at 1 year. There was no difference between cohorts who had received different doses of THY. Similarly, nonrelapse mortality was not affected by plasma BU levels. Overall 17 patients have died, including 5 with disease progression/relapse, at 19 to 262 days (median, 75 days) after transplantation. Other than relapse, the major causes of death were organ toxicity and infections.

Table 3. Causes of Death⁎
Cause of DeathRegimen
tBUCY/THYtBUCY
RelatedURDRelatedURD
Relapse23——
Pulmonary
Pneumonia——1—
DAD/MOF————
Aspergillus—111
Viral2———
Respiratory failure/ARDS/IPS13—1
MOF—2——
VOD1———
Cardiac——2—
GVHD11—1
Suicide——1—
Total71054

ARDS indicates acute respiratory distress syndrome; DAD, diffuse alveolar damage; GVHD, graft-versus-host disease; IPS, interstitial pneumonitis syndrome; MOF, multiorgan failure; tBUCY, targeted busulfan + cyclophosphamide; THY, Thymoglobulin; URD, unrelated donor; VOD, veno-occlusive disease of the liver (sinusoidal obstruction syndrome).

⁎ With follow-up of 12 to 42 months.

Among concurrent controls conditioned with tBUCY, the nonrelapse mortality was 3.7% at day 100 and 22% at 1 year. Overall, 9 patients have died, 75 to 639 days (median, 255 days) after HCT. Other than relapse (related to advanced disease stage in patients conditioned with tBUCY + THY), there were no significant differences in causes of death between tBUCY + THY–treated patients and concurrent controls.

Viral Reactivation 

Epstein-Barr virus 

An increase in the plasma titer of EBV DNA to >1000 copies per milliliter occurred in 1 patient (given 6 mg/kg of THY). There was no evidence of a lymphoproliferative disorder. The patient was treated successfully with a single dose of 375 mg/m2 of the anti-CD20 antibody rituximab as previously described by Carpenter et al. [29].

Cytomegalovirus 

CMV reactivation tended to occur earlier in patients who received THY; however, overall only 18 (32%) of 56 patients reactivated (CMV antigenemia), compared with 15 (56%) of 27 concurrent controls who did not receive THY (Figure 2). The proportions of patients with ≥10 positive cells per slide (200 000 cells) were comparable in the 2 groups (4/56 [7%] and 2/27 [7%], respectively). Whereas only 1 seronegative patient with a seronegative donor became CMV positive (a tBUCY-conditioned control patient), reactivation after transplantations for which the donor, the patient, or both were CMV seropositive occurred in 18 (45%) of 40 tBUCY + THY–conditioned patients and 14 (78%) of 18 concurrent controls. Three patients at high risk for CMV reactivation, 2 treated with tBUCY + THY (days 14 and 16) and 1 treated with tBUCY only (day 444), developed CMV disease.

  • View full-size image.
  • Figure 2. 

    Reactivation of CMV. Shown is the cumulative incidence of CMV antigenemia in patients conditioned with tBUCY + THY and tBUCY (concurrent controls). Low risk: donor and patient were seronegative; high risk: donor, patient, or both were seropositive.

Other Infections 

During the first 100 days after HCT, bacteremias occurred in 42% of patients who received THY and in 60% of concurrent controls not given THY. There was no difference in the spectrum of bacteria identified between the 2 groups.

Invasive fungal infections were documented in 3 patients in each group. In tBUCY + THY–treated patients, Aspergillus species were documented at 1.5 to 4.5 months. One patient died from fungal pneumonia, 1 from problems associated with renal failure, and 1 from encephalitis and GVHD. In concurrent controls who had not received THY, Aspergillus species were documented in 2 (at 3 and 11 months), and Candida glabrata, Aspergillus fumigatus, and Rhizopus species were found in 1 patient (at 2 months). Two patients died with aspergillus pneumonia, and 1 is surviving.

Effect of Graft Composition on Relapse, GVHD, and Survival 

Data on the phenotypic composition of the cells collected from the transplant donors are summarized in Table 4. There was a slightly lower proportion of CD3+ cells in the transplant inoculum for patients conditioned with tBUCY + THY than in concurrent controls (P = .07). However, there was no recognizable effect of the content of CD34+, CD3+, CD4+, CD8+, or CD56+ cells on the incidence of acute GVHD or nonrelapse mortality. There was a suggestion, however, that higher numbers of CD3+/CD56+ donor cells were correlated with a lower incidence of relapse in patients conditioned with tBUCY and THY (hazard ratio, 0.3; P = .07).

Table 4. Graft Composition
Cell TypetBUCY/THYtBUCY
CD34+ (106/kg)7.2(0.1-30.3)7.6(3.2-17.2)
CD3+ (108/kg)2.0(0.2-5.8)2.5(0.3-4.4)
CD3+/CD56+ (106/kg)11.3(0.2-28.0)12.0(2.8-59.2)

Data are median (range) of cells infused.

Comorbid Conditions and Transplantation Outcomes 

Because comorbid conditions may have a major effect on posttransplantation outcome [32], we also analyzed results on the basis of comorbidity scores. The comorbidity score as modified by Sorror et al. [33] was 0 to 7 (median, 1) for tBUCY + THY–conditioned patients and 0 to 4 (median, 1) for the tBUCY-conditioned concurrent controls. The hazard ratio for nonrelapse mortality was higher for patients with comorbidity scores of ≥3 (1.7; confidence interval, 0.6-4.5), but this effect did not reach significance (P = .28).

Comparison of Results with Historical Controls 

The concurrent control group of patients who did not receive THY consisted of good-risk patients with a very low relapse risk. Therefore, we also compared results in tBUCY + THY–conditioned patients with those in a historical control cohort conditioned with tBUCY and composed of patients with good-risk MDS (similar to concurrent controls) and high-risk MDS (comparable to tBUCY + THY–conditioned patients). Data on relapse-free survival, relapse, and GVHD are summarized in Table 5. Overall, the data suggest that the addition of THY to a tBUCY regimen had a beneficial effect on GVHD (in particular, chronic GVHD; Figure 3) and, with current follow-up, did not increase the incidence of disease recurrence as compared with historical controls (Figure 4A) and resulted in comparable relapse-free survival (Figure 4B). Because these data were not derived from a randomized study, comparisons must be made with great caution. This point is further underscored by the fact that patients in this study received PBPCs as a source of stem cells, whereas historical controls underwent transplantation with marrow. As illustrated in Figure 3, the incidence of chronic GVHD in patients who underwent transplantation with PBPCs after conditioning with tBUCY + THY was comparable to that observed in historical controls who were not given THY but who received bone marrow as a source of stem cells.

Table 5. Outcome in Subgroups of Patients Conditioned with tBUCY+ THY (Current Study) and in Concurrent and Historical Controls
End PointtBUCY/THY (Current)tBUCY (Concurrent Controls)tBUCY (Historical Controls)
RA/RARS (n)NA⁎2757
RFSNA78%72%
RelapseNA0%5%
Acute GVHDNA82%67%
Chronic GVHDNA82%40%
RAEB (n)14—20
RFS55%NA60%
Relapse35%NA20%
Acute GVHD35%NA75%
Chronic GVHD42%NA50%
AML (n)17—10
RFS58%NA40%
Relapse24%NA30%
Acute GVHD50%NA50%
Chronic GVHD15%NA30%
Advanced disease (n)†56—36
RFS56%NA56%
Relapse21%NA19%
Acute GVHD50%NA64%
Chronic GVHD34%NA47%

AML indicates acute myelogenous leukemia, de novo or evolving from MDS; RA, refractory anemia; RAEB, RA with excess blasts; RARS, RA with ringed sideroblasts; RFS, relapse-free survival; tBUCY, targeted busulfan + cyclophosphamide; THY, Thymoglobulin; NA, not applicable.

⁎ Four patients in this study had <5% marrow myeloblasts but high-risk cytogenetics and were included under “Advanced Disease.”

† All patients conditioned with tBUCY/THY in this study were considered as having advanced disease (including 4 patients with <5% marrow blasts but with high-risk cytogenetics).

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  • Figure 3. 

    Chronic GVHD in tBUCY + THY–conditioned patients and in concurrent and historical controls (conditioned with tBUCY). Shown are the cumulative incidences in tBUCY + THY–treated patients who received the prescribed doses of THY (PBPC, THY, 4.5 and 6.0) and tBUCY-conditioned concurrent (PBPC, concurrent) and historical controls (Bone Marrow, historical controls).

  • View full-size image.
  • Figure 4. 

    Outcome in patients conditioned with tBUCY + THY (this study) and in historical controls conditioned with tBUCY without THY. A, Cumulative incidence of relapse. B, Relapse-free survival.

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Discussion 

This study shows that THY at doses of 4.5 to 6 mg/kg can be used safely in combination with BU and CY to condition patients for allogeneic HCT. However, 15% of patients did not receive the prescribed dose of THY because of clinically significant adverse reactions or patient and attending physician preferences. Because the study protocol did not allow for further THY dose escalation if a patient experienced EBV reactivation, the maximum THY dose reached was 6 mg/kg. The cumulative incidence rates of acute and chronic GVHD for all evaluable patients were 50% and 34%, respectively. Although the reported incidence rates of acute GVHD, particularly for grades I and II, have shown considerable variation, comparison to results in concurrent controls conditioned with tBUCY only, without the addition of THY, suggested a beneficial effect of THY. Such an effect seemed to be even stronger for chronic GVHD, for which incidence rates have shown greater consistency over time [34]. Although the objective of this study was the determination of an optimal THY dose, we observed no correlation of THY dose and incidence of GVHD, although serum levels correlated significantly with THY doses administered. These observations would be consistent with the concept that the most effective THY levels that could be achieved with the current regimen were achieved with the lowest study dose (4.5 mg/kg). Reassuring was the fact that, with the current follow-up, there was no increase in the incidence of relapse in comparison to historical controls conditioned with tBUCY. Also, although EBV reactivation occurred in a single patient, overall there was no evidence for an increase in viral reactivation in comparison to patients not given THY. In fact, CMV reactivation occurred in a smaller proportion of THY-treated patients than among concurrent controls not given THY. This pattern was, presumably, related to the lower incidence of GVHD and, hence, the less frequent use of glucocorticoids in THY-treated patients.

In contrast to historical controls, patients in this study, with 1 exception, received G-CSF–mobilized PBPCs, which have been associated with an increased incidence of GVHD, particularly in its chronic form [35]. Against this background, the current results suggest that the use of THY interfered with the development of chronic GVHD and maintained the GVHD incidence at a level previously observed with the use of bone marrow as the stem cell source [1]. An interaction between PBPC and THY has also been suggested by other investigators [36].

In an earlier study, Russell et al. [14] at the University of Calgary Transplant Center reported an incidence of grades II to IV acute GVHD of 8% with related and 19% with unrelated donors in patients with various diagnoses given THY (4.5 mg/kg) as part of the conditioning regimen. Nonrelapse mortality was 5% by day 100, compared with 12.5% in this study. However, the studies are difficult to compare. The Calgary regimen used fludarabine and BU instead of BUCY for transplantation conditioning. Fludarabine is thought to be associated with less nonhematologic toxicity than CY, and it is conceivable that prevention of chemotherapy-induced damage to potential target organs of GVHD, such as the liver and intestinal tract, resulted in a lower incidence or fewer clinical manifestations of GVHD. This is consistent with the concept that the regimen intensity correlates with the development of GVHD [8, 9].

Bacigalupo et al. [37] conducted 2 randomized THY studies that enrolled 109 patients with various diagnoses who underwent transplantation from unrelated donors and compared THY 7.5 and 15 mg/kg with patients who did not receive THY. The incidence of GVHD with the lower dose of THY was comparable to that observed in controls. Among patients given THY 15 mg/kg, only 11% developed grades III and IV acute GVHD, compared with 50% in controls (P = .001). However, the use of the higher dose of THY was complicated by a high rate of fatal infections (30% versus 7% in controls; P = .02). Similar to the current trial, chronic GVHD developed in 39% of patients who received THY (at any dose), compared with 62% in controls (P = .04). As in this study, there was no improvement in overall or relapse-free survival in THY-treated patients.

Remberger et al. [12] reported a matched cohort study of unrelated transplant recipients, 52 of whom were conditioned with 10 Gy of single-dose total body irradiation (TBI) plus CY and 10 mg/kg THY given on days −5 to −1 (Huddinge) and 104 of whom were conditioned with 12 to 14.4 Gy of fractionated TBI and not given THY (Seattle). THY-treated patients had a lower incidence of nonrelapse mortality (P = .005) and overall mortality (P = .03) than patients not given THY. Relapse rates were not different. The Huddinge team then presented results in 162 patients with hematologic malignancies who were conditioned with BUCY or CY/TBI and received transplants from unrelated donors. Patients received 4, 6, 8, or 10 mg/kg THY in addition to CSP and methotrexate as GVHD prophylaxis. The incidence rates of acute GVHD grades II to IV (III/IV) were 29% (15%), 32% (6%), 16% (5%), and 17% (2%) for the 4 dose levels of THY, respectively. The corresponding rates for chronic GVHD were 41%, 18%, 46%, and 51%, respectively. Nonrelapse mortality for the entire group was 11% at day 100 and was 17%, 16%, 28%, and 25% (P = .03) at 3 years for the 4 THY dose levels, respectively. Patients given 6 mg/kg THY had the highest probability of survival. Although the underlying diagnoses varied somewhat among THY dose groups, the results suggest that increasing the THY dose beyond 6 mg/kg had no net beneficial effect on outcomes.

The day 100 nonrelapse mortality of 12.5% in patients conditioned with tBUCY + THY in the present study was somewhat higher than the 5% reported by the Calgary team, but it was comparable to the results in the Huddinge study and to our own historical data [1]. Considering the limited numbers of patients in the studies under consideration, conclusions must be drawn with caution.

Also of interest in this study was the observation that the dose of CD3+/CD56+ cells correlated inversely with the probability of relapse (P = .07). However, the study was not designed to further investigate such an effect or determine a possible interaction of THY with CD3+/CD56+ cells. Finally, the hazard rate for relapse was 3.5 for patients with steady-state BU levels <800 ng/mL compared with higher levels, although this difference was not significant (P = .17). An effect of BU levels on relapse would be consistent with observations in patients with chronic myeloid leukemia in whom lower steady-state BU levels were associated with an increased incidence of disease recurrence after transplantation [16].

Taken together, these data and those reported by others [11, 13, 14] suggest that THY at dosages of 4.5 to 8 mg/kg may reduce the incidence of both acute and chronic GVHD after unrelated, and probably related, donor transplantation. Of concern is the observation that some 15% to 20% of patients showed poor tolerance of THY, although adverse reactions have also been observed with other preparations of antithymocyte globulin. Review of the clinical records also suggests that, with increasing experience with THY, premedications can be adjusted such that the drug is tolerated by most patients.

It is not clear why, despite a reduced incidence of GVHD and no increase in the relapse probability, relapse-free survival was not improved. In part this may be due to limited patient numbers. The observation also raises the question of whether the results obtained by Russell et al. [14] were related not only to the use of THY, but also to the combination of THY with fludarabine (rather than CY) and BU. Combinations of fludarabine/BU have yielded very encouraging results even without the addition of THY. The M.D. Anderson group, for example, observed a treatment-related 1-year mortality of 3% among 96 patients with AML or MDS who were conditioned with fludarabine and intravenous BU and received transplants from related or unrelated donors [2]. At our center, we observed a 7% day 100 mortality in an earlier study in patients with high-risk MDS conditioned with fludarabine and oral BU [38]. These data suggest that combinations of THY with fludarabine/BU regimens deserve to be further explored. The results underscore the need for prospective randomized trials.

In summary, the present study suggests that incorporation of THY in a tBUCY conditioning regimen may be beneficial in reducing the incidence of GVHD. So far, this has not caused prohibitive adverse effects on relapse or infectious complications. Although only a very limited dose finding was possible in this study, available data suggest an optimal dose of THY in the range of 4.5 to 8 mg/kg. These observations warrant further study.

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Acknowledgments 

This work was supported in part by National Institutes of Health (Bethesda, MD) grant nos. HL36444, CA18029, CA15704, and HL66947. We thank our patients who agreed to participate in these studies and all physicians, nurses, and support staff involved in their care. We thank Joanne Greene and Elizabeth Soll for maintaining the patient database, Linda Reiser for the determination of busulfan levels, Eileen Bryant, PhD, for cytogenetic analysis, Kieren Marr for the data on fungal infections, and Bonnie Larson, Helen Crawford, and Sue Carbonneau for manuscript preparation. We thank the Genzyme Corporation for support in general and the determination of Thymoglobulin levels in particular.

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PII: S1083-8791(05)01417-5

doi:10.1016/j.bbmt.2005.12.036

Biology of Blood and Marrow Transplantation
Volume 12, Issue 5 , Pages 573-584, May 2006

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