Previous randomized graft-versus-host disease (GVHD)-prophylaxis trials have failed to demonstrate reduced incidence and severity of chronic GVHD (cGVHD). Here we reanalyzed and updated a randomized phase 3 trial comparing standard GVHD prophylaxis with or without pretransplantation ATG-Fresenius (ATG-F) in 201 adult patients receiving myeloablative conditioning before transplantation from unrelated donors. The cumulative incidence of extensive cGVHD after 3 years was 12.2% in the ATG-F group versus 45.0% in the control group (P < .0001). The 3-year cumulative incidence of relapse and of nonrelapse mortality was 32.6% and 19.4% in the ATG-F group and 28.2% and 33.5% in the control group (hazard ratio [HR] = 1.21, P = .47, and HR = 0.68, P = .18), respectively. This nonsignificant reduction in nonrelapse mortality without increased relapse risk led to an overall survival rate after 3 years of 55.2% in the ATG-F group and 43.3% in the control group (HR = 0.84, P = .39, nonsignificant). The HR for receiving immunosuppressive therapy (IST) was 0.31 after ATG-F (P < .0001), and the 3-year probability of survival free of IST was 52.9% and 16.9% in the ATG-F versus control, respectively. The addition of ATG-F to standard cyclosporine, methotrexate GVHD prophylaxis lowers the incidence and severity of cGVHD, and the risk of receiving IST without raising the relapse rate. ATG-F prophylaxis reduces cGVHD morbidity.

Allogeneic hematopoietic stem cell transplantation (HSCT) is increasingly used worldwide as a curative therapy for malignant and nonmalignant hematologic disorders. Chronic graft-versus-host disease (cGVHD) is the leading cause of nontransplantation mortality and morbidity after allogeneic HSCT.1-3  cGVHD is a multiorgan disease resembling autoimmune disorders, such as scleroderma or systemic lupus.4,5  Its incidence and prevalence are rising because of transplantation practices known to be associated with increased risk of cGVHD.4,6  Indeed, older patients now undergo HSCT, and more transplantations are being performed from unrelated donors and/or with peripheral blood stem cells instead of bone marrow. Furthermore, the reduced-intensity conditioning (RIC) regimens developed during recent years have also led to higher numbers of transplantations performed worldwide.7,8  However, although the acute GVHD (aGVHD) rate appears lower after RIC, the incidence of cGVHD seems to be unaffected.9  Altogether, cGVHD thus remains the most challenging complication after allogeneic HSCT.10 

The main risk factor for developing cGVHD is the previous occurrence of aGVHD.11  Thus, transplantation physicians have focused on decreasing the rate of aGVHD to lower nonrelapse mortality (NRM) associated with both aGVHD and cGVHD. However, although calcineurin inhibitors (cyclosporine or tacrolimus) in association with methotrexate have proven to decrease the aGVHD rate in randomized studies and although new regimens, such as the association of rapamycin with tacrolimus, seem to lower the aGVHD rate in phase 2 trials, none of those prophylactic regimens has reduced the incidence rate and severity of cGVHD.12-14 

A key pathophysiologic role of donor T cells in the initiation and development of cGVHD is recognized.4  Donor T cells are also main effectors of aGVHD.4  Based on these experimental data, T-cell depletion has been proposed as a tool to lower GVHD incidence and severity. However, whereas numerous phase 2 trials have been conducted,15  few randomized trials using T-cell depletion versus drug prophylaxis have been performed.16-20  Lessons from all but one21  of these randomized trials indicated that, although aGVHD might be reduced and the relapse rate increased, cGVHD incidence rate and severity remained basically unchanged after T-cell depletion.

Different types of anti–T-cell globulin (ATG) preparations have been tested as part of conditioning regimens to achieve in vivo T-cell depletion so as to prevent GVHD.22  Anti-Jurkat ATG-Fresenius (ATG-F), in addition to cyclosporine and methotrexate, has shown promising results in several phase 2 trials for transplantation from matched or mismatched unrelated donors.23-25  We previously reported that, adding ATG-F to standard cyclosporine, methotrexate GVHD prophylaxis significantly reduced severe acute in a randomized phase 3 trial.26  The cumulative incidence of aGVHD grade 3 and 4 was 11.7% in the ATG-F group versus 25.5% in the control group (adjusted hazard ratio [HR] = 0.48, 95% confidence interval [CI], 0.24-0.96, P = .039), and cumulative incidence of grade 2 and 4 was 33.0% in the ATG-F group versus 52.0% in the control group (adjusted HR = 0.55, 95% CI, 0.35-0.85, P = .008). These results slightly differ from results published by Finke et al26  because one patient in the control group originally classified as having aGVHD grade 1 was later reviewed again and classified as grade 3. Here we present final data and unpublished results on cGVHD with extended follow-up.

Study design

We previously reported this randomized, multicenter, open-label, phase 3 trial to compare standard GVHD prophylaxis plus pretransplantation ATG-F to standard GVHD prophylaxis alone (control) in adult patients receiving myeloablative conditioning before hematopoietic cell transplantation from matched unrelated donors.26  All patients received myeloablative conditioning regimens containing total body irradiation (8-12 Gy), or busulfan (14-16 mg/kg orally or equivalent for intravenous administration) plus cyclophosphamide (2 × 60 mg/kg) or etoposide; or regimens containing thiotepa ≥ 15 mg/kg or carmustine ≥ 300 mg/m2. All patients received cyclosporine starting on day −1 with target trough levels ≥ 200 ng/mL in combination with methotrexate 15 mg/m2 on day 1 and 10 mg/m2 on days 3, 6, and 11. Cyclosporine was recommended to be tapered after day 100. Patients in the ATG-F group received additional ATG-F at a dose of 20 mg/kg on day −3, day −2, and day −1 (total dose, 60 mg/kg) before transplantation. Concerning cGVHD, primary physicians were asked to stage organ involvement by severity (none, mild, moderate, and severe) and then to grade patients according to Shulman et al as none, limited, or severe.27  Other details on regimen and patients population are summarized in the supplemental Methods (available on the Blood Web site; see the Supplemental Materials link at the top of the online article).

All patients gave written informed consent in accordance with the Declaration of the Helsinki, and approval was given by the University Medical Center Freiburg Institutional Review Board. Project management, statistical planning and analysis, randomization, data management, and clinical monitoring were conducted by the Clinical Trials Center, University Medical Center Freiburg, Freiburg, Germany. Patients were followed until 2 years after recruitment of the last patient, as specified in the study protocol.

Prespecified endpoints included incidence and severity of aGVHD, incidence and severity of cGVHD (cGVHD, limited/extensive and extensive), incidence of relapse, incidence of NRM (defined as death without preceding relapse), disease-free survival, overall survival, infections, and adverse events. Extensive versus limited cGVHD was defined according to Seattle's group criteria.27  However, aGVHD beyond day 100 in the absence of cGVHD was categorized as late aGVHD in accordance with the guidelines of the National Institutes of Health.28  In case acute and chronic symptoms of GVHD were present simultaneously, the GVHD was classified as cGVHD.26 

Statistical analysis

Treatment groups were compared with respect to time to cGVHD (limited/extensive and extensive), time to cGVHD in different target organs (skin, eyes, mouth, lung, liver), time to relapse, time to NRM, overall survival time, time under immunosuppressive therapy (IST), and time to late bacterial infections (after day 100). Additional definitions of parameters are provided in the supplemental Methods.

Treatment groups with respect to time-to-event variables were compared with Cox regression models for the event-specific hazard functions using 2-sided Wald tests. Disease status (early vs advanced) and stem-cell source (bone marrow vs peripheral blood) were included for adjustment. To estimate the effect size, the HR of ATG-F versus control was calculated with 95% CI. In addition to the cumulative incidence rates29  as estimators of probability of event over time, we calculated Cox model-based rates adjusted for the covariates disease status and stem-cell source.30,31 

The effect of cGVHD (limited/extensive and extensive) on relapse and NRM was investigated with a Cox regression model with cGVHD as time-dependent covariate. The HRs of cGVHD versus no cGVHD were estimated with 95% CI, and cumulative hazard functions were displayed for illustration.

Treatment groups were compared with respect to the transition hazards between the states “alive and free of IST” and “alive under IST” using a Cox regression model, including disease status and stem-cell source for adjustment. Transition HRs were estimated with 95% CI using robust estimators of SEs.32  The probabilities of survival under IST and of survival free of IST (adding up to the overall survival probability) over time were estimated with the Aalen-Johansen estimator33  using the R package “etm.”34 

Statistical analysis was performed using the Statistical Analysis System, Version 9.2, and R, Version 2.11.1. The study is registered with WHO primary registers at www.clinicaltrials.gov as #DRKS00000002 and at https://drksneu.uniklinite-freiburg.del/drks_web as #NCT00655343.

Study patients

The patient population has already been described in detail.26  A total of 202 patients were randomized in 31 centers. One patient was not transplanted. A total of 201 patients (103 ATG-F, 98 control patients) with median age of 40 years (range, 18-60 years), transplanted between 2003 and 2007, with acute myeloid leukemia (n = 101), myelodysplastic syndrome (n = 10), acute lymphoid leukemia (n = 70), chronic myeloid leukemia (n = 17), osteomyelofibrosis (n = 3) in early (first complete response or myelodysplastic syndrome-refractory anemia, n = 107), or advanced status of disease (all other, n = 94) were observed for a median follow-up time of 3 years (25% quartile, 2.5; 75% quartile, 3.9 years). Patient, disease, and transplantation characteristics are summarized in Table 1.

Table 1

Baseline patient characteristics

CharacteristicATG-F* (N = 103)Control (N = 98)
Patient age (median, range) 40 (18-60) 39 (18-60) 
    < 40 y 47 50 
    ≥ 40 y 56 48 
Donor age*(median, range) 35 (20-58) 37 (18-56) 
    < 40 y 62 64 
    ≥ 40 y 32 30 
Patient sex   
    Male 58 58 
    Female 45 40 
Patient/donor sex*   
    Patient male/donor female 14 13 
    Other 87 85 
Patient/donor CMV status   
    Negative/negative 23 44 
    Negative/positive 14 16 
    Positive/negative 32 19 
    Positive/positive 34 19 
HLA-C mismatch*   
    Yes 21 14 
    No 75 75 
Type of disease   
    Acute lymphoid leukemia 37 33 
    Acute myeloid leukemia 55 46 
    Chronic myeloid leukemia 11 
    Myelodysplastic syndrome 
    Osteomyelofibrosis 
Disease status   
    Early 64 43 
    Advanced 39 55 
Conditioning regimen   
    Total body irradiation/cyclophosphamide 54 48 
    Busulfan/cyclophosphamide 26 26 
    Total body irradiation/etoposide/cyclophosphamide 11 
    Total body irradiation/other 
    No total body irradiation/other 
Stem cell source   
    Bone marrow 21 16 
    Peripheral blood 82 82 
CharacteristicATG-F* (N = 103)Control (N = 98)
Patient age (median, range) 40 (18-60) 39 (18-60) 
    < 40 y 47 50 
    ≥ 40 y 56 48 
Donor age*(median, range) 35 (20-58) 37 (18-56) 
    < 40 y 62 64 
    ≥ 40 y 32 30 
Patient sex   
    Male 58 58 
    Female 45 40 
Patient/donor sex*   
    Patient male/donor female 14 13 
    Other 87 85 
Patient/donor CMV status   
    Negative/negative 23 44 
    Negative/positive 14 16 
    Positive/negative 32 19 
    Positive/positive 34 19 
HLA-C mismatch*   
    Yes 21 14 
    No 75 75 
Type of disease   
    Acute lymphoid leukemia 37 33 
    Acute myeloid leukemia 55 46 
    Chronic myeloid leukemia 11 
    Myelodysplastic syndrome 
    Osteomyelofibrosis 
Disease status   
    Early 64 43 
    Advanced 39 55 
Conditioning regimen   
    Total body irradiation/cyclophosphamide 54 48 
    Busulfan/cyclophosphamide 26 26 
    Total body irradiation/etoposide/cyclophosphamide 11 
    Total body irradiation/other 
    No total body irradiation/other 
Stem cell source   
    Bone marrow 21 16 
    Peripheral blood 82 82 

Data are N or median (range).

HLA-C indicates human leukocyte antigen locus C.

*

Donor age unknown for 13 donors (ATG-F, N = 9; control, N = 4), donor sex unknown for 2 donors (ATG-F, N = 2; control, N = 0), and HLA-C mismatch unknown for 17 patients (ATG-F, N = 7; control, N = 9).

cGVHD incidence and severity

Of the 170 patients alive without second transplantation at day 100 (ATG-F, n = 90; control, n = 80), 75 patients experienced cGVHD (limited or extensive) (ATG-F, n = 27; control, n = 48), with 47 patients presenting extensive cGVHD (ATG-F, n = 11; control, n = 36). Detailed patient presentation is provided as supplemental Table 1. The cumulative incidence of extensive cGVHD after 3 years was 12.2% in the ATG-F group versus 45.0% in the control group (HR = 0.20, 95% CI, 0.10-0.39, P < .0001, Figure 1A). Cumulative incidence of limited/extensive cGVHD was 30.0% and 60.0% in the ATG-F versus control, respectively (HR = 0.34, 95% CI, 0.21-0.55, P < .0001). Treatment effect was also assessed in different subgroups of patients defined by prognostic factors (Figure 1B). ATG-F significantly reduced cGVHD (defined as limited/extensive or extensive alone) whatever the patient sex, patient or donor age, type of disease, disease status, stem cell source, or patient cytomegalovirus status was. Compared with the results shown in Finke et al26  (supplemental Methods), Figure 1B now shows slightly different, but similar, results after a median follow-up time of 3 years. The cumulative incidence of late aGVHD after 3 years was 4.4% in the ATG-F group versus 11.3% in the control group.

Figure 1

Cumulative incidence of extensive chronic GVHD by treatment groups overall and by prognostic subgroups. (A) Effect of treatment on extensive cGVHD. (B) Treatment effects within prognostic subgroups with regard to cGVHD, analyzed with Cox regression models adjusted for disease status and stem-cell source.

Figure 1

Cumulative incidence of extensive chronic GVHD by treatment groups overall and by prognostic subgroups. (A) Effect of treatment on extensive cGVHD. (B) Treatment effects within prognostic subgroups with regard to cGVHD, analyzed with Cox regression models adjusted for disease status and stem-cell source.

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We then analyzed how ATG-F prophylaxis affected cGVHD in different target organs. As shown in supplemental Table 1 and supplemental Figure 2, cumulative incidences were reduced in all main cGVHD target organs: skin (3-year cumulative incidence, 5.6% vs 27.0%; ATG-F vs control, HR = 0.18, 95% CI, 0.07-0.48, P = .0006), eyes (2.2% vs 20.7%; HR = 0.10, 95% CI, 0.02-0.45, P = .0025), mouth (4.4% vs 18.8%; HR = 0.24, 95% CI, 0.08-0.74, P = .013), lung (3.3% vs 16.3%; HR = 0.17, 95% CI, 0.05-0.61, P = .006), and liver (1.1% vs 21.2%; HR = 0.05, 95% CI, 0.01-0.39, P = .004). The incidences in other, less involved target organs were not calculated because of few events, but crude numbers are described in a footnote to supplemental Table 1.

Impact of cGVHD on relapse rate and NRM

cGVHD lowered the relapse rate, resulting in an HR of 0.50 (95% CI, 0.26-0.96, P = .037; Figure 2A). Of note, GVHD prophylaxis with ATG-F was not associated with increased risk of relapse because the 3-year cumulative incidence of relapse was 32.6% in the ATG-F group and 28.2% in the control group (shown in supplemental Figure 3B; HR = 1.21, 95% CI, 0.72-2.02, P = .47). There was a trend to an increase in NRM by extensive cGVHD, resulting in an HR of 2.06 (95% CI, 0.93-4.58, P = .075; Figure 2B); the 3-year cumulative incidence of NRM was 19.4% in the ATG-F group and 33.5% in the control group (shown as supplemental Figure 3C; HR = 0.68, 95% CI, 0.38-1.20, P = .18). This still nonsignificant reduction in NRM without increased relapse risk led to an overall survival after 3 years of 55.2% in the ATG-F group and 43.3% in the control group (HR = 0.84, 95% CI, 0.56-1.25, P = .39; Figure 3A), and similar disease-free survival in both groups (supplemental Figure 3A).

Figure 2

Effect of chronic GVHD on relapse and nonrelapse mortality. (A) Effect of cGVHD (limited and extensive) on relapse effect estimated from Cox model with time-dependent covariate (HR = 0.49, 95% CI, 0.26-0.96, P = .037). (B) Effect of cGVHD (extensive) on NRM. Effect estimated from Cox model with time-dependent covariate (HR = 2.06, 95% CI, 0.93-4.58, P = .075).

Figure 2

Effect of chronic GVHD on relapse and nonrelapse mortality. (A) Effect of cGVHD (limited and extensive) on relapse effect estimated from Cox model with time-dependent covariate (HR = 0.49, 95% CI, 0.26-0.96, P = .037). (B) Effect of cGVHD (extensive) on NRM. Effect estimated from Cox model with time-dependent covariate (HR = 2.06, 95% CI, 0.93-4.58, P = .075).

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Figure 3

Split of overall survival probability into probability of survival free of IST and probability of survival under IST. (A) Overall survival probability. (B) Probability of survival free of IST. (C) Probability of survival under IST.

Figure 3

Split of overall survival probability into probability of survival free of IST and probability of survival under IST. (A) Overall survival probability. (B) Probability of survival free of IST. (C) Probability of survival under IST.

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Effect of ATG-F on treatment duration and time to stop IST

The HR for receiving IST was 0.31 (95% CI, 0.18-0.55, P < .0001), and that for being able to stop IST was 2.02 (95% CI, 1.41-2.91, P = .0001, ATG-F vs control, respectively). At 3 years, the probability of being alive and free of IST was 52.9% and 16.9%, and that of being alive and still under IST was 2.4% and 26.3% in the ATG-F versus control, respectively (Figure 3B-C). As can be seen in Figure 3, the probabilities of being alive and free of IST and of being alive under IST add up to the probability of overall survival. Although we see only a slight survival advantage of ATG-F versus control, our results show that patients in the ATG-F group predominantly live free of IST whereas patients in the control group predominantly live under IST.

Effect of ATG-F on late bacterial infection and cause of late nonrelapse-related death

We previously reported the occurrence of 4 fatal cases of post-transplantation lymphoproliferative disorders in patients treated in the ATG-F arm. In the present paper aiming to study cGVHD, we focus our attention to late bacterial infections, which are the main cause of mortality and lead to significant morbidity in patients with cGVHD.4  We analyzed here the distribution of these infectious complications. Main bacterial species were Streptococcus, Staphylococcus, Klebsiella, and Pseudomonas species (Table 2). The main infectious locations were bloodstream (n = 38, 17 vs 21), urinary tract (n = 19, 15 vs 4), oral/gastrointestinal (n = 18, 10 vs 8), and pulmonary (n = 18, 6 vs 12; Table 2) in the ATG-F group versus control group, respectively. The 3-year cumulative incidence of late bacterial infection (after day 100) was 26.3% versus 38.8% (Figure 4) in the ATG-F group versus control group, respectively (HR = 0.68, 95% CI, 0.39-1.17, P = .16). Main causes of non–relapse-related deaths over the long-term (> 100 days) included infection (5 and 4 patients) and cGVHD (0 and 4 patients), in the ATG-F group versus control group, respectively (Table 2). Overall updated causes of death are summarized in supplemental Table 2.

Table 2

Late bacterial infection (> day 100, 121 infections occurring in 54 patients: 25 ATG-F, 29 control)

SpeciesNLocation: bloodstream/pneumonia/urinary/otherMedian elapsed time (range)ATG-F/control
Streptococcal 29 6/2/10/11 315 (120-1479) 17/12 
Staphylococcal 22 13/5/0/4 223 (105-1164) 9/13 
Klebsiella 11 3/1/4/3 227 (103-431) 8/3 
Pseudomonas 16 9/0/0/7 190 (118-418) 2/14 
Other specified 20 6/4/3/7 292 (107-1390) 9/11 
Other nonspecified 23 10/6/2/5 176 (101-1385) 12/11 
All 121 47/18/19/37 245 (101-1479) 57/64 
SpeciesNLocation: bloodstream/pneumonia/urinary/otherMedian elapsed time (range)ATG-F/control
Streptococcal 29 6/2/10/11 315 (120-1479) 17/12 
Staphylococcal 22 13/5/0/4 223 (105-1164) 9/13 
Klebsiella 11 3/1/4/3 227 (103-431) 8/3 
Pseudomonas 16 9/0/0/7 190 (118-418) 2/14 
Other specified 20 6/4/3/7 292 (107-1390) 9/11 
Other nonspecified 23 10/6/2/5 176 (101-1385) 12/11 
All 121 47/18/19/37 245 (101-1479) 57/64 

Infectious-related deaths after day 100: ATG-F arm: 5 deaths: streptococcal sepsis (1), sepsis, not otherwise specified (2), CMV disease (1), and influenzae pneumonia (1). Of those 5 deaths, the 2 sepsis occurred in context of cGVHD. Control arm: 4 deaths: toxoplasma (1), Pseudomonas species (1), aspergillosis (1), and legionella (1). All 4 of those deaths occurred in context of cGVHD.

Figure 4

Effect of treatment on late bacterial infection. Treatment effect estimated from Cox model adjusted for disease status and stem-cell source (HR [ATG-F vs control]= 0.68, 95% CI, 0.39-1.17, P = .16).

Figure 4

Effect of treatment on late bacterial infection. Treatment effect estimated from Cox model adjusted for disease status and stem-cell source (HR [ATG-F vs control]= 0.68, 95% CI, 0.39-1.17, P = .16).

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Prognostic factors for developing cGVHD

We finally aimed to study prognostic factors for developing cGVHD in our randomized trial (supplemental Table 4). Cox regression analyses of prognostic factors for developing extensive cGVHD adjusted for treatment arm and aGVHD (as a time-dependent factor) revealed 2 factors associated with increased extensive cGVHD risk: donor age of 40 years or more (HR = 2.01, 95% CI, 1.08-3.72, P = .027) and type of disease (HRs = 3.88, 95% CI, 1.25-12.0, 1.56, 0.76-3.20, and 2.63, 1.12-6.16 for patients with myelodysplastic syndrome, acute lymphoid leukemia, and chronic myeloid leukemia/osteomyelofibrosis compared with acute myeloid leukemia, respectively; P = .039; supplemental Table 4A). Factors found to be associated with increased cGVHD risk (limited or extensive) were type of disease (HRs = 1.88, 95% CI, 0.75-4.73, 1.60, 0.88-2.90, and 2.78, 1.38-5.58, for patients with myelodysplastic syndrome, acute lymphoid leukemia, and chronic myeloid leukemia/osteomyelofibrosis compared with acute myeloid leukemia, respectively; P = .023) and a non–irradiation-based conditioning regimen (HR = 2.58, 95% CI, 1.49-4.46, P = .0007; supplemental Table 4B).

We present here data on cGVHD with extended follow-up (median, 3 years) on the largest randomized trial comparing standard GVHD prophylaxis versus standard prophylaxis plus ATG. The addition of ATG-F to cyclosporine, methotrexate GVHD prophylaxis significantly reduces the incidence and severity of cGVHD and the time to treat patients with IST without increasing relapse rate. In addition, our data showed that not only incidence, but disease severity and organ involvement, strongly favors the use of ATG-F. These data demonstrate that ATG-F prophylaxis decreases cGVHD morbidity.

Although ex vivo T-cell depletion has been used for decades with the aim to reduce aGVHD, there have been very few randomized trials.15-20  Indeed, one randomized study of hematopoietic cell transplantation from human leukocyte antigen-identical sibling donors already in the late 1980s demonstrated a decrease in the incidence of aGVHD, no difference in cGVHD, and an increased risk of relapse and rejection.16,17  Furthermore, T-cell depletion has been associated with increased risk of post-transplantation lymphoproliferative disorder.15-20  This risk of post-transplantation lymphoproliferative disorder was also found in our trial with 4 fatal cases in the ATG-F arm compared with no case in the control group. Only 2 randomized trials have been conducted on the use of ATG or T-cell depletion in hematopoietic cell transplantation from unrelated donors.18,19  An American trial17  compared 405 patients receiving bone marrow from unrelated donors, 2 different in vitro T-cell depletion methods (by anti–T-cell monoclonal antibody T10B9 [MEDI-500, Medimmune] or counter flow elutriation in combination with in vivo use of equine antithymocyte globulin [Pharmacia/Pfizer] and cyclosporine to a control group receiving cyclosporine and short-course methotrexate alone). The experimental group presented less severe aGVHD, but T-cell depletion had no influence on the incidence of cGVHD or survival.17  In the Italian sequential trial19,21  testing in vivo T-cell depletion, a total of 109 patients received bone marrow from unrelated donors and standard GVHD prophylaxis with or without antithymocyte globulin (Thymoglobulin, Genzyme). The reduction in GVHD was accompanied by a higher risk for lethal infections resulting in no improvement in survival. Extensive cGVHD developed more frequently in patients not given ATG.20  In a retrospective, nonrandomized analysis, the French registry35  most recently assessed the impact of rabbit ATG, incorporated within a standard myeloablative conditioning regimen before allogeneic stem cell transplantation using unrelated donors. In their retrospective series of 120 patients with leukemia, 69 did not receive ATG, whereas 51 patients did. With a median follow-up of 30.3 months, the cumulative incidence of extensive cGVHD was significantly lower in the ATG group compared with the “no-ATG” group (4% vs 32%, respectively; P = .0017). In multivariate analysis, the absence of ATG use was the strongest parameter associated with increased risk of extensive cGVHD (RR = 7.14; 95% CI, 1.7-33.3, P = .008). At 2 years, the probability of NRM, relapse, and overall and leukemia-free survivals did not significantly differ between the “no-ATG” and “ATG” groups. Thus, our trial definitively proves results suggested by the French retrospective analysis, as well as data suggested by the Italian sequential trials on a large patient population mainly receiving peripheral blood stem cells.

cGVHD is associated with a strong antileukemic effect,4-6  as confirmed here because cGVHD was associated with a nearly 50% decrease in relapse risk. However, in contrast with other reports using T-cell depletion showing increased propensity to relapse,15  GVHD prophylaxis with ATG-F was not associated with increased risk of relapse. However, the study was not powered to detect differences in relapse risk among the 2 groups.

In addition to this graft-versus-leukemia effect, there was a trend that extensive cGVHD also raised NRM in our cohort, as reported by others.1,3,10,36-39  However, while decreasing 3-year NRM incidence by > 10% (on an absolute scale), ATG-F did not lead to a large improvement in overall survival on a relative scale (ATG-F 55.2% vs control 43.3%). Improved supportive care and antimicrobial therapies of patients with cGVHD40  most probably explain why decreased incidence and severity of cGVHD in the ATG-F arm do not yet translate to improved survival. Similarly, although the risk of severe bacterial infections was higher in the control group, as expected from cGVHD rates, neither the late bacterial infection rate nor bacterial-related deaths significantly differs between the 2 treatment groups.

Finally, as discussed elsewhere, the most stringent proof of any prophylactic or treatment efficacy in cGVHD is to compare its ability to provide quality-of-life-improved survival without immunosuppressive drug therapy between 2 treatment groups.37,41-44  Indeed, the cGVHD course is typified by flare and remission episodes, and the withdrawal of any drugs to attain immune tolerance is the ultimate goal in treating this disease. Our data demonstrate that patients in the ATG-F group had a 100% better chance than the controls of being able to stop IST. At 3 years, the ATG-F group's probability of being alive and free of IST was 3-fold higher than that of the control group.

Prognostic factors for developing cGVHD after adjusting for previous aGVHD and ATG-F treatment showed that patients with acute myelogenous leukemia had a slightly lower risk of developing cGVHD and that older donor's age and the use of a non–irradiation-based conditioning regimen raised the risk. We have no formal explanations for those results. These results must be confirmed because multiple comparisons were performed.

However, it needs to be made very clear that (1) these data do not necessarily apply to other brands of ATG, and (2) that they do apply only to ablative HSCT. Furthermore, in a recent retrospective Center for International Blood and Marrow Transplant Research analysis of RIC HSCT using, in a nonrandomized setting, other ATG brands showed worse outcomes with ATG.45  Thus, our results may not apply to RIC HSCT. Finally, most of the stem cell products used in our study were grafts from peripheral blood, although there were minorities of marrow infusions: thus, our data may not apply to marrow grafts in which there is a lower incidence of cGVHD.

In conclusion, the addition of ATG-F to standard GVHD prophylaxis significantly reduces the incidence, severity, and morbidity of cGVHD and the risk of receiving IST. It does not increase the risk of relapse and may ultimately provide a long-term survival advantage.

An Inside Blood analysis of this article appears at the front of this issue.

The online version of this article contains a data supplement.

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 USC section 1734.

The authors thank Jan Beyersmann and Anika Buchholz, Institute of Medical Biometry and Medical Informatics, University Medical Center, Freiburg, Germany, for support of the statistical analysis, and Carole Cuerten, Department of Hematology and Oncology, University Medical Center, Freiburg, Germany, for editorial assistance.

The study was supported by Fresenius Biotech GmbH, Germany.

Contribution: G.S., W.A.B., H.D.O., M.S., A.R.Z., L.V., T.R., D.A.H., R.S., K.K., J.M., J.A.M., W.L., E.H., V.K., M.B., H.E., H.-J.K., H.B., M.E., and J.F. contributed to the recruitment and treatment of patients in the trial; J.F., C.S., O.G., and G.S. contributed to the design and analysis of the trial; and all authors contributed to the interpretation of the data and saw and approved the final version of the manuscript.

A complete list of the ATG-Fresenius Trial Group members appears in the “Appendix.”

Conflict-of-interest disclosure: J.F. has received travel and lecture fees from Fresenius Biotech GmbH. W.A.B. has received consultancy and lecture fees from Fresenius Biotech GmbH. K.K. has received travel and lecture fees from Fresenius Biotech GmbH. H.B. has received travel and lecture fees from Fresenius Biotech GmbH. M.E. has received travel fees from Fresenius Biotech GmbH. The other authors declare no competing financial interests.

Correspondence: Gérard Socié, Hematology/Transplantation, Hospital Saint Louis & University paris VII, 1 Avenue Claude vellefaux 75475 Paris, Cedex 10, France; e-mail: [email protected].

1
Socie
 
G
Stone
 
JV
Wingard
 
JR
et al. 
Long-term survival and late deaths after allogeneic bone marrow transplantation: Late Effects Working Committee of the International Bone Marrow Transplant Registry.
N Engl J Med
1999
, vol. 
341
 
1
(pg. 
14
-
21
)
2
Bhatia
 
S
Francisco
 
L
Carter
 
A
et al. 
Late mortality after allogeneic hematopoietic cell transplantation and functional status of long-term survivors: report from the Bone Marrow Transplant Survivor Study.
Blood
2007
, vol. 
110
 
10
(pg. 
3784
-
3792
)
3
Martin
 
PJ
Counts
 
GW
Appelbaum
 
FR
et al. 
Life expectancy in patients surviving more than 5 years after hematopoietic cell transplantation.
J Clin Oncol
2010
, vol. 
28
 
6
(pg. 
1011
-
1016
)
4
Lee
 
SJ
Vogelsang
 
G
Flowers
 
ME
Chronic graft-versus-host disease.
Biol Blood Marrow Transplant
2003
, vol. 
9
 
4
(pg. 
215
-
233
)
5
Bhushan
 
V
Collins
 
RH
Chronic graft-vs-host disease.
JAMA
2003
, vol. 
290
 
19
(pg. 
2599
-
2603
)
6
Vogelsang
 
GB
How I treat chronic graft-versus-host disease.
Blood
2001
, vol. 
97
 
5
(pg. 
1196
-
1201
)
7
Gratwohl
 
A
Baldomero
 
H
Aljurf
 
M
et al. 
Hematopoietic stem cell transplantation: a global perspective.
JAMA
2010
, vol. 
303
 
16
(pg. 
1617
-
1624
)
8
Bertz
 
H
Potthoff
 
K
Finke
 
J
Allogeneic stem-cell transplantation from related and unrelated donors in older patients with myeloid leukemia.
J Clin Oncol
2003
, vol. 
21
 
8
(pg. 
1480
-
1484
)
9
Mielcarek
 
M
Martin
 
PJ
Leisenring
 
W
et al. 
Graft-versus-host disease after nonmyeloablative versus conventional hematopoietic stem cell transplantation.
Blood
2003
, vol. 
102
 
2
(pg. 
756
-
762
)
10
Socie
 
G
Salooja
 
N
Cohen
 
A
et al. 
Nonmalignant late effects after allogeneic stem cell transplantation.
Blood
2003
, vol. 
101
 
9
(pg. 
3373
-
3385
)
11
Atkinson
 
K
Horowitz
 
MM
Gale
 
RP
et al. 
Risk factors for chronic graft-versus-host disease after HLA-identical sibling bone marrow transplantation.
Blood
1990
, vol. 
75
 
12
(pg. 
2459
-
2464
)
12
Deeg
 
HJ
How I treat refractory acute GVHD.
Blood
2007
, vol. 
109
 
10
(pg. 
4119
-
4126
)
13
Antin
 
JH
Kim
 
HT
Cutler
 
C
et al. 
Sirolimus, tacrolimus, and low-dose methotrexate for graft-versus-host disease prophylaxis in mismatched related donor or unrelated donor transplantation.
Blood
2003
, vol. 
102
 
5
(pg. 
1601
-
1605
)
14
Cutler
 
C
Li
 
S
Ho
 
VT
et al. 
Extended follow-up of methotrexate-free immunosuppression using sirolimus and tacrolimus in related and unrelated donor peripheral blood stem cell transplantation.
Blood
2007
, vol. 
109
 
7
(pg. 
3108
-
3114
)
15
Ho
 
VT
Soiffer
 
RJ
The history and future of T-cell depletion as graft-versus-host disease prophylaxis for allogeneic hematopoietic stem cell transplantation.
Blood
2001
, vol. 
98
 
12
(pg. 
3192
-
3204
)
16
Maraninchi
 
D
Gluckman
 
E
Blaise
 
D
et al. 
Impact of T-cell depletion on outcome of allogeneic bone-marrow transplantation for standard-risk leukaemias.
Lancet
1987
, vol. 
2
 
8552
(pg. 
175
-
178
)
17
Blaise
 
D
Gravis
 
G
Maraninchi
 
D
Long-term follow-up of T-cell depletion for bone marrow transplantation.
Lancet
1993
, vol. 
341
 
8836
(pg. 
51
-
52
)
18
Wagner
 
JE
Thompson
 
JS
Carter
 
SL
Kernan
 
NA
Effect of graft-versus-host disease prophylaxis on 3-year disease-free survival in recipients of unrelated donor bone marrow (T-cell Depletion Trial): a multi-centre, randomised phase II-III trial.
Lancet
2005
, vol. 
366
 
9487
(pg. 
733
-
741
)
19
Bacigalupo
 
A
Lamparelli
 
T
Bruzzi
 
P
et al. 
Antithymocyte globulin for graft-versus-host disease prophylaxis in transplants from unrelated donors: 2 randomized studies from Gruppo Italiano Trapianti Midollo Osseo (GITMO).
Blood
2001
, vol. 
98
 
10
(pg. 
2942
-
2947
)
20
Pavletic
 
SZ
Carter
 
SL
Kernan
 
NA
et al. 
Influence of T-cell depletion on chronic graft-versus-host disease: results of a multicenter randomized trial in unrelated marrow donor transplantation.
Blood
2005
, vol. 
106
 
9
(pg. 
3308
-
3313
)
21
Bacigalupo
 
A
Lamparelli
 
T
Barisione
 
G
et al. 
Thymoglobulin prevents chronic graft-versus-host disease, chronic lung dysfunction, and late transplant-related mortality: long-term follow-up of a randomized trial in patients undergoing unrelated donor transplantation.
Biol Blood Marrow Transplant
2006
, vol. 
12
 
5
(pg. 
560
-
565
)
22
Mohty
 
M
Mechanisms of action of antithymocyte globulin: T-cell depletion and beyond.
Leukemia
2007
, vol. 
21
 
7
(pg. 
1387
-
1394
)
23
Finke
 
J
Schmoor
 
C
Lang
 
H
Potthoff
 
K
Bertz
 
H
Matched and mismatched allogeneic stem-cell transplantation from unrelated donors using combined graft-versus-host disease prophylaxis including rabbit anti-T lymphocyte globulin.
J Clin Oncol
2003
, vol. 
21
 
3
(pg. 
506
-
513
)
24
Schleuning
 
M
Gunther
 
W
Tischer
 
J
Ledderose
 
G
Kolb
 
HJ
Dose-dependent effects of in vivo antithymocyte globulin during conditioning for allogeneic bone marrow transplantation from unrelated donors in patients with chronic phase CML.
Bone Marrow Transplant
2003
, vol. 
32
 
3
(pg. 
243
-
250
)
25
Zander
 
AR
Kroger
 
N
Schleuning
 
M
et al. 
ATG as part of the conditioning regimen reduces transplant-related mortality (TRM) and improves overall survival after unrelated stem cell transplantation in patients with chronic myelogenous leukemia (CML).
Bone Marrow Transplant
2003
, vol. 
32
 
4
(pg. 
355
-
361
)
26
Finke
 
J
Bethge
 
WA
Schmoor
 
C
et al. 
Standard graft-versus-host disease prophylaxis with or without anti-T-cell globulin in haematopoietic cell transplantation from matched unrelated donors: a randomised, open-label, multicentre phase 3 trial.
Lancet Oncol
2009
, vol. 
10
 
9
(pg. 
855
-
864
)
27
Shulman
 
HM
Sullivan
 
KM
Weiden
 
PL
et al. 
Chronic graft-versus-host syndrome in man: a long-term clinicopathologic study of 20 Seattle patients.
Am J Med
1980
, vol. 
69
 
2
(pg. 
204
-
217
)
28
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.
Biol Blood Marrow Transplant
2005
, vol. 
11
 
12
(pg. 
945
-
956
)
29
Gooley
 
TA
Leisenring
 
W
Crowley
 
J
Storer
 
BE
Estimation of failure probabilities in the presence of competing risks: new representations of old estimators.
Stat Med
1999
, vol. 
18
 
6
(pg. 
695
-
706
)
30
Makuch
 
RW
Adjusted survival curve estimation using covariates.
J Chronic Dis
1982
, vol. 
35
 
6
(pg. 
437
-
443
)
31
Rosthoj
 
S
Andersen
 
PK
Abildstrom
 
SZ
SAS macros for estimation of the cumulative incidence functions based on a Cox regression model for competing risks survival data.
Comput Methods Programs Biomed
2004
, vol. 
74
 
1
(pg. 
69
-
75
)
32
Lin
 
DY
Wei
 
LJ
The robust inference for the proportional hazards model.
J Am Stat Assoc
1989
, vol. 
84
 (pg. 
1074
-
1078
)
33
Andersen
 
PK
Borgan
 
O
Gill
 
RD
Keiding
 
N
Statistical Models Based on Counting Processes
1993
New York, NY
Springer-Verlag
34
Allignol
 
A
Schumacher
 
M
Beyersmann
 
J
Empirical transition matrix of multistate models: the ETM package.
J Stat Software
2011
, vol. 
7
 
13
(pg. 
1
-
15
)
35
Mohty
 
M
Labopin
 
M
Balère
 
ML
et al. 
Antithymocyte globulins and chronic graft-versus-host disease after myeloablative allogeneic stem cell transplantation from HLA-matched unrelated donors: a report from the Societé Française de Greffe de Moelle et de Therapie Cellulaire.
Leukemia
2010
, vol. 
24
 
11
(pg. 
1867
-
1874
)
36
Lee
 
SJ
Klein
 
JP
Barrett
 
AJ
et al. 
Severity of chronic graft-versus-host disease: association with treatment-related mortality and relapse.
Blood
2002
, vol. 
100
 
2
(pg. 
406
-
414
)
37
Stewart
 
BL
Storer
 
B
Storek
 
J
et al. 
Duration of immunosuppressive treatment for chronic graft-versus-host disease.
Blood
2004
, vol. 
104
 
12
(pg. 
3501
-
3506
)
38
Sullivan
 
KM
Agura
 
E
Anasetti
 
C
et al. 
Chronic graft-versus-host disease and other late complications of bone marrow transplantation.
Semin Hematol
1991
, vol. 
28
 
3
(pg. 
250
-
259
)
39
Yakoub-Agha
 
I
Mesnil
 
F
Kuentz
 
M
et al. 
Allogeneic marrow stem-cell transplantation from human leukocyte antigen-identical siblings versus human leukocyte antigen-allelic-matched unrelated donors (10/10) in patients with standard-risk hematologic malignancy: a prospective study from the French Society of Bone Marrow Transplantation and Cell Therapy.
J Clin Oncol
2006
, vol. 
24
 
36
(pg. 
5695
-
5702
)
40
Couriel
 
D
Carpenter
 
PA
Cutler
 
C
et al. 
Ancillary therapy and supportive care of chronic graft-versus-host disease: National Institutes of Health Consensus Development Project on Criteria for Clinical Trials in Chronic Graft-Versus-Host Disease: V. Ancillary Therapy and Supportive Care Working Group Report.
Biol Blood Marrow Transplant
2006
, vol. 
12
 
4
(pg. 
375
-
396
)
41
Socie
 
G
Ritz
 
J
Martin
 
PJ
Current challenges in chronic graft-versus-host disease.
Biol Blood Marrow Transplant
2010
, vol. 
16
 
1 suppl
(pg. 
S146
-
S151
)
42
Sorror
 
ML
Leisenring
 
W
Deeg
 
HJ
Martin
 
PJ
Storb
 
R
Twenty-year follow-up of a controlled trial comparing a combination of methotrexate plus cyclosporine with cyclosporine alone for prophylaxis of graft-versus-host disease in patients administered HLA-identical marrow grafts for leukemia.
Biol Blood Marrow Transplant
2005
, vol. 
11
 
10
(pg. 
814
-
815
)
43
Griffith
 
LM
Pavletic
 
SZ
Lee
 
SJ
Martin
 
PJ
Schultz
 
KR
Vogelsang
 
GB
Chronic graft-versus-host disease: implementation of the National Institutes of Health Consensus Criteria for Clinical Trials.
Biol Blood Marrow Transplant
2008
, vol. 
14
 
4
(pg. 
379
-
384
)
44
Martin
 
PJ
Weisdorf
 
D
Przepiorka
 
D
et al. 
National Institutes of Health Consensus Development Project on Criteria for Clinical Trials in Chronic Graft-versus-Host Disease: VI. Design of Clinical Trials Working Group report.
Biol Blood Marrow Transplant
2006
, vol. 
12
 
5
(pg. 
491
-
505
)
45
Soiffer
 
R
LeRademacher
 
J
Ho
 
VT
et al. 
Impact of in vivo T-cell depletion on outcome of reduced intensity conditioning hematopoietic cell transplantation for hematologic malignancies [Abstract].
Blood
2010
, vol. 
116
 
21
pg. 
951
 
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