Comparison of Two Doses of Antithymocyte Globulin in Patients ...

Biology of Blood and Marrow Transplantation 14:913-919 (2008) Q 2008 American Society for Blood and Marrow Transplantation 1083-8791/08/1408-0001$32.00/0 doi:10.1016/j.bbmt.2008.05.023

Comparison of Two Doses of Antithymocyte Globulin in Patients Undergoing Matched Unrelated Donor Allogeneic Stem Cell Transplantation Francis Ayuk, Galina Diyachenko, Tatjana Zabelina, Christine Wolschke, Boris Fehse, Ulrike Bacher, Rudolf Erttmann, Nicolaus Kro¨ger, Axel R. Zander Department of Stem Cell Transplantation, University Medical Center Hamburg, Hamburg, Germany Correspondence and reprint requests to: Francis Ayuk, MD, Stem Cell Transplantation, University Hospital Hamburg-Eppendorf, Martinistrasse 52, D-20246 Hamburg, Germany (e-mail: [email protected]). Received March 23, 2008; accepted May 28, 2008

ABSTRACT Antithymocyte globulin (ATG) as part of conditioning regimens is known to reduce the incidence and severity of acute and chronic graft-versus-host disease (aGVHD, cGVHD). The influence of ATG on transplant-related mortality (TRM) and disease-free survival (DFS) is controversial, and may depend on the dose and timing of ATG. We retrospectively compared 2 doses of ATG-Fresenius (ATG-F) in patients undergoing matched unrelated donor allogeneic hematopoetic stem cell transplantation (HSCT) for hematologic malignancies. A dose of 60 mg/kg body weight has previously been recommended for ATG-F. All patients received cyclosporine A and short course methotrexate. ATG-F was administered at a dose of 30 mg/kg on day –1 (ATG-30 group, n 5 34) or 20 mg/kg/day on days –3 to –1 (ATG-60 group, n 5 49). There was no difference in time to leukocyte and platelet engraftment in the 2 groups. The incidence of aGVHD grade II-IV (50% versus 53%, P 5 .83) and grade III-IV (27 versus 20%, P 5 .60) was similar in the ATG-30 versus ATG-60 groups, respectively. There was a trend to a higher incidence of cGVHD in the ATG-30 group (59% versus 40%, P 5 .14). The estimated 3-year incidence of relapse was similar in the ATG-30 and ATG-60 groups (15% versus 16%, P 5 .84) whereas the 2-year TRM was lower for the ATG-30 group (12% versus 33%, P 5 0.02), mainly because of a higher incidence of fatal infections in the ATG-60 group. This resulted in a better DFS (73% versus 51%, P 5 .07) for the ATG-30 group. ATG-F (30 mg/kg) administered as a single dose on day –1 may lead to better outcome in patients undergoing unrelated donor allogeneic HSCT compared to 60 mg/kg given in 3 equivalent doses. A prospective randomized study comparing these 2 doses of ATG-F is warranted.

Ó 2008 American Society for Blood and Marrow Transplantation

KEY WORDS ATG Dose Stem cell transplantation

INTRODUCTION Allogeneic hematopoetic stem cell transplantation (HSCT) has over the past decades become the treatment of choice for many hematologic malignant and nonmalignant diseases [1,2]. Although better established [3,4], transplantion from an HLA-identical sibling is only available for about 1/3 of patients. The use of stem cell grafts from matched unrelated donors is associated with higher risk of complications and lower survival rates compared to matched sibling donors [5-9] for reasons that may include undetected HLA disparities [10,11]. One of the key complications of unrelated donor transplantation is graft-versus-host disease (GVHD) [5-11]. Several strategies have been

developed to prevent severe GVHD, including ex vivo T cell depletion [12,13], in vivo T cell depletion with antithymocyte globulin (ATG) [14-17], or alemtuzumab [18], as well as the use of posttransplant combined ciclosporine A or FK506-based immunosuppression [19]. Inclusion of ATG in conditioning regimens for allogeneic stem cell transplantation (SCT) is associated with reduced risk of acute and chronic GVHD (aGVHD, cGVHD) and may also improve overall survival (OS) rates [20]. Presently there are 3 commercially available ATG products: ATGAMÒ (Pharmacia, Piscataway, NJ) is produced by immunizing horses with human thymocytes. ThymoglobulinÒ (Genzyme, 913

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Cambridge, MA) is produced by immunizing rabbits with human thymocytes, whereas ATG-FreseniusÒ (ATG-F, Fresenius, Germany) is produced by immunizing rabbits with the jurkat cell line. ATG is usually administered on the days preceeding stem cell infusion in single or multiple doses. The different ATG preparations listed above possess differential immunomodulatory potencies and are thus used in different doses. For a given ATG preparation the observed effect may not only be dependent on the dose applied but also on timing of the application [21]. ATG administered prior to transplantation can still be detected in patients in the days after transplantation [22]. The mechanism of immunosuppresion induced by ATG may depend on the concentration attained in vivo and includes T cell depletion, modulation of adhesion and trafficking molecules, depletion and modulation of dendritic cells, as well as induction of regulatory T cells [23]. Although very low doses may lose the immunosuppressive effects, very high doses of ATG may aggravate the delay in immune recovery and increase the risk of infections [24-27]. The recommended dose for Thymoglobulin is 6-8 mg/kg body weight (BW) [24]. For ATG-F, a total dose of 60 mg/kg BW is recommended [28]. It is important to note that the recommended doses of ATG are based on a few studies; thus, further investigation is required to established optimal dosing and timing. In the present study we compare 2 doses of ATG-F (30 mg/kg versus 60 mg/kg BW) administered as part of conditioning regimen for allogeneic SCT from matched unrelated donors. PATIENTS AND METHODS Between July 1997 and October 2005, 83 adult and pediatric patients with hematologic malignancies were treated with bone marrow or blood stem cell grafts from an unrelated donor. GVHD prophylaxis consisted of ATG, methotrexate, and cyclosporine A. The conditioning regimen consisted of total-body irradiation (TBI) and alkylating agents or a combined alkylating therapy. Patients’ characteristics are listed in Table 1. Patients with CML in first chronic phase, AML in first or second complete remission, ALL in first or second complete remission, and MDS (RA and RARS) were classified as ‘‘good-risk’’ patients. All other patients were classified as ‘‘bad risk.’’ Follow-up only includes surviving patients. Donor Selection and HLA Typing

HLA-A and HLA-B antigen loci were typed using the reverse SSO line-blot assay. HLA-DRB1 and HLA-DQB1 loci were typed using the reverse SSO dotblot method. Helmberg-Score software was used for allelic assignment in conjunction with the most recent nomenclature report and library [29-31]. Remaining ambiguities were resolved by sequencing

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Table 1. Patients’ and Donors’ Characteristics

Characteristic

ATG-30 n 5 34

ATG-60 n 5 49

Patient age Median 37 (r: 1-61) 35 (r: 1-52) Donor age Median 36 (r: 21-51) 33 (r: 21-58) Patient sex Male 14 (41%) 26 (53%) Female 20 (59%) 23 (47%) Donor sex Male 17 (50%) 37 (75%) Female 17 (50%) 12 (25%) Donor/patient sex match Sex matched 19 (56%) 32 (65%) Sex mismatched 15 (44%) 17 (35%) Graft source Bone marrow 14 (41%) 17 (35%) PBSC 20 (59%) 32 (65%) Diagnosis CML 7 (21%) 15 (31%) ALL 7 (21%) 20 (41%) AML, sAML 17 (50%) 7 (14%) MDS, sMDS, CMML 3 (8%) 7 (14%) Risk Good risk 22 (65%) 35 (71%) Bad risk 12 (35%) 14 (29%) Transplant period 1997-2002 14 (41%) 18 (37%) 2003-2005 20 (59%) 31 (63%) CMV (patient) Seropositive 15 (44%) 23 (47%) Seronegative 19 (56%) 26 (53%) CMV (donor) Seropositive 11 (32%) 26 (53%) Seronegative 23 (68%) 23 (47%) CD 341 cells x 106/kg Median 5.9 (r: 1.2-16.8) 5.8 (r: 0.8-27.7) Conditioning (Busulfan versus TBI) Busulfan 24 (71%) 28 (57%) TBI 10 (29%) 21 (43%) Conditioning with 23 (68%) 29 (59%) Etoposide

PValue .30 .79 .4

.02

.5

.7

.005

.63

.82

.8

.08

.84 .3

.5

TBI indicates total-body irridation; ATG, antithymocyte globulin; CMV, cytomegalovirus; AML, acute myelogenous leukemia; ALL, acute lymphoblastic leukemia; MDS, myelodysplastic syndromes; PBSC, peripheral blood progenitor cells.

of amplimers obtained after SSP with appropriate primers. HLA-A and B were resolved at the 2-digit level, whereas HLA-DRB1 and DQB1 were typed to the 4-digit allelic level. Typing for HLA-C was not routinely performed and disparities, if observed, were not considered as mismatch. Conditioning Regimens

Patients with CML received busulfan (14–16 mg/ kg BW orally) and cyclophosphamide (2 60 mg/ kg/BW) or TBI (12 Gy, divided into 2 Gy fractions twice daily) and cyclophosphamide (2 60 mg/kg/ BW). Patients with AML and MDS received busulfan

Two Doses of Antithymocyte Globulin in Allogeneic Stem Cell Transplantation

(14-16 mg/kg BW), etoposide (30–45 mg/kg BW), and cyclophosphamide (2 60 mg/kg/BW). Patients with ALL received TBI (12 Gy, divided into 2 Gy fractions twice daily), cyclophosphamide (2 60 mg/kg/BW), and etoposide (45 mg/kg BW). Conditioning regimens were balanced in the 2 groups. GVHD Prophylaxis and Management

All patients received cyclosporine A (3 mg/kg i.v.), starting on day 21 and additional short-course methotrexate (10 mg/m2) on days 1, 3, and 6 after transplantation. ATG-F was administered at a dose of 30 mg/kg on day –1 (ATG-30 group) or 20 mg/kg/day on days – 3 to –1 (ATG-60 group). Initially (1997) all patients received 30 mg/kg of ATG-F. Because of the notion at that time that higher doses may be of advantage, we increased the dose of ATG and eventually adopted the proposed standard dose of 60 mg/kg. In an interimanalysis we noticed that the ATG-30 group had been doing very well so we reintroduced this lower dose of ATG in 2002. Acute GVHD was treated with methylprednisolone (2 mg/kg). Second-line treatment included tacrolimus (instead of cyclosporine A), mycophenolat mophetil and basiliximab.

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any cause other than recurrent malignancy. DFS refers to patients alive without relapse. The probability of OS and DEF were calculated using the method of Kaplan-Meier. Comparison of groups was performed using the log-rank test. Cumulative incidence of TRM and relapse were calculated using a Gray-test [34] (according to NHMRC Clinical Trials Centre, University of Sydney, 2007, Prof. Val Gebski), taking into account competing risks TRM and relapse. The following factors were included in the analysis (only pretransplant parameters): patients’ age, donors’ age, patients’ sex, donors’ sex, diagnosis, good-risk versus bad-risk disease, CMV status of patient, CMV status of donor, stem cell source (bone marrow versus peripheral blood stem cells), TBI versus busulfan-based conditioning, etoposide versus nonetoposide conditioning, number of transplanted CD341 cells/kg BW, sex mismatch versus matched, period of transplant (1997-2002 versus 2003-2005), and ATG dose. Variables for which a P-value \.1 was found were then entered into a multivariate Cox regression model. RESULTS Engraftment

Grading of GVHD

The grading of aGVHD was performed according to standard criteria. The grades were assigned on the severity of aGVHD of the skin, liver, and gastrointestinal tract [32]. Chronic GVHD was evaluated in patients alive beyond day 180 posttransplant and characterized as ‘‘limited’’ or ‘‘extensive’’ [33].

One patient in the ATG-60 group died before engraftment. In all, no graft failure was observed. There was no difference in time to leukocyte engraftment for the ATG-30 (15 days) and ATG-60 (16 days) groups, P 5 .16. Time to platelet engraftment was 20 days in both groups, P 5 .85 (Table 2). GVHD

Supportive Care

All patients were nursed in reverse isolation in a conventional or laminar air flow room. Acyclovir and fluconazol or itraconazol were routinely administed to all patients. Prophylaxis against Pneumocystis carinii consisted of cotrimoxazole or pentamidine inhalation. All blood products were irradiated with 25 Gy. CMV-negative patients received only CMVnegative blood products. CMV-positive patients were monitored at least weekly for CMV infection by PCR and/or antigenaemia assay. Preemptive therapy was started with 10 mg/kg gancyclovir per day after 2 consecutive positive PCR results or 1 positive antigenaemia assay. Neutropenic fever was treated with broad-spectrum antibiotics. Statistical Considerations

All analyses were performed with the SPSS version 10/11 (SPSS Inc., Chicago, IL). Endpoints of the analysis were transplant-related mortality (TRM), incidence, and severity of aGVHD, incidence and severity of cGVHD, incidence of relapse, diseasefree survival (DFS) and OS. TRM is defined as from

All 83 patients were evaluable for aGVHD, whereas 67 patients were evaluable for cGVHD. The incidence of aGVHD grade II-IV (50% versus 53%, P 5 .83) and grade III-IV (27 versus 20%, P 5 .60) was similar in the ATG-30 versus ATG-60 groups, respectively. There was a trend to a higher incidence of cGVHD in the ATG-30 group (59 versus 40 %, P 5 .14). Infections and Other Causes of Death

Overall, 9 patients in the ATG-30 group and 21 patients in the ATG-60 group died. Thirteen of 21 (62%) deaths in the ATG-60 and 3 of the 9 deaths (33%) in the ATG-30 group were associated with infections with or without GVHD (Table 3). Routine monitoring for viraemia was performed for CMV. No cases of EBV-associated posttransplant lymphoproliferative disorders were observed. The incidence of CMV reactivation was similar in the ATG-30 and ATG-60 groups (27% versus 26%). Four of 21 deaths (19%) in the ATG-60 group and 1 of 9 (11%) in the ATG-30 group were associated with viral infections. Eight deaths (38%) in the ATG-60 group and 2

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Table 2. Clinical Outcomes According to ATG Dose ATG-30 n 5 34 Graft-failure Engraftment (day) median (range) Leukocytes $1.0 109/L Platelets $20 109/L Acute GVHD Grade II-IV Grade III-IV Chronic GVHD Overall Limited Extensive TRM (at 2 years)* Relapse (at 3 years)* OS (at 3 years)† DFS (at 5 years)† Follow-up, median days (range)

ATG-60 n 5 49

0/34 (0%)

P-Value

0/48 (0%)

15 (11-28) 20 (10-60)

1.0

16 (9-28) 20 (10-130)

.16 .85

17/34 (50%) 9/34 (27%)

26/49 (53%) 10/49 (20%)

.83 .60

17/29 (59%) 10/29 (35%) 7/29 (24%) 12% (95% CI:1%-23%) 15% (95% CI: 3%-27%) 72% (95%CI: 57%-87%) 73% (95%CI:62%-88%) 658 (188-3114)

15/38 (40%) 8/38 (21%) 7/38 (19%) 33% (95% CI: 20%-46%) 16% (95% CI: 5%-27%) 56% (95%CI: 42%-70%) 51% (95%CI:36%-66%) 850 (192-1389)

.14

.02 .84 .12 .07 .90

OS indicates overall survival; DFS, disease-free survival; TRM, transplant-related mortality, ATG, antithymocyte globulin; GVHD, graftversus-host disease. *Gray-test. †Log-rank test.

(22%) in the ATG-30 group were associated with fungal infections. Five deaths (24%) in the ATG-60 group and 5 (56%) in the ATG-30 group were because of relapse. OS

The estimated 3-year OS was 72% for patients in the ATG-30 group and 56% for those in the ATG-60 group, P 5 .12 (Figure 1). None of the relevant parameters had significant influence on OS in the univariate analysis.

Treatment-Related Mortality

The 2-year cumulative incidence of treatment-related mortality (using the Gray-test for competing risks) was lower for the ATG-30 group (12%) compared to the ATG-60 group (33%), P 5 .02 (Figure 3). In the univariate analysis period of transplant 20022005 was also associated with lower treatment-related mortality (p 5 .08). In the multivariate analysis, both ATG dose 60 mg/kg (RR: 3.5, P 5 .03) and transplant period 1997-2002 (RR: 2.5, P 5 .045) retained their negative influence on treatment-related mortality.

DFS

There was a trend to better estimated 3-year DFS for the ATG-30 group with 73% compared to 51% for the ATG-60 group, P 5 .07 (Figure 2). In the univariate analysis, ATG dose 60 mg/kg (relative risk [RR]: 2.02, P 5 .08) and patient’s CMV-seropositivity (RR: 1.97, P 5 .06) had negative impact on outcome. In the multivariate analysis, both ATG dose of 60 mg/ kg (RR: 1.9, P 5 .09) and patient’s CMV-seropositivity (RR: 1.9, P 5 .09) retained their negative impact on DFS (Table 4.).

Relapse

The 3-year cumulative incidence of relapse (using Gray-test for competing risks) was similar in the ATG-30 and ATG-60 groups (15% versus 16%), P 5 .84 (Figure 4). Bad-risk disease (RR: 4.0, P 5 .046) was the only factor with significant impact on relapse. Conditioning with etoposide was associated with a trend to higher relapse incidence in the univariate analysis

Table 4. Multivariate Proportional Hazard Regression Analysis of All Patients Table 3. Causes of Death According to ATG Dose Cause of death

ATG-30

ATG-60

Infection 1 GVHD Infection Relapse Toxicity Others Total

1 (11.1%) 2 (22.2%) 5 (55.6%) 1 (11.1%) 0 (0%) 9 (100%)

5 (23.8%) 8 (38.1%) 5 (23.8%) 2 (9.5%) 1 (4.8%) 21 (100%)

ATG indicates antithymocyte globulin; GVHD, graft-versus-host disease.

Relative Confidence PRisk Interval (95 % CI) Value DFS ATG dose 60 mg/kg Patient CMV-seropositive TRM ATG dose 60 mg/kg Transplant year 1997-2002

1.9 1.9

0.90-4.21 0.94-3.86

.09 .08

3.5 2.5

1.03-9.30 1.02-6.00

.03 .045

DFS indicates disease-free survival; TRM, transplant-related mortality; ATG, antithymocyte globulin; CMV, cytomegalovirus.


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probability of overall survival

1,0

0,8

ATG 30 mg/kg

0,6

ATG 60 mg/kg

0,4

0,2

0,0 0

500

1000

1500

2000

2500

3000

3500

days after transplantation Figure 3. Represents cumulative estimates of TRM using the Graytest for competing risks.

Figure 1. Represents Kaplan-Meier estimates of OS.

(P 5 .07). However, it must be taken into consideration that all patients with ALL and AML but only 29% of patients with CML received etoposide. This is of relevance because the 3-year relapse rates for patients with ALL, AML, and CML were 29%, 25%, and 9%, respectively. DISCUSSION

probability of disease-free survival

The dose and timing of ATG may have significant impact on outcome of patients undergoing allogeneic SCT from unrelated donors. The positive impact of ATG on the incidence of aGVHD and cGVHD is well documented [20]; however, the effects on treatment-related mortalizy and DFS remain controversial [14-17,35] and may depend on the dose and timing as well as on the brand of ATG used. For Thymoglobulin, doses between 4.5 and 8 mg/kg BW have been recommended, depending on timing [17,24,36]. A retrospective analysis in CML patients undergoing matched unrelated donor allografting reported

better OS and DFS for patients receiving $60 mg/kg BW of ATG-F compared to \60 mg/kg BW. This was in part because of a higher incidence of severe acute GVHD in the group with \60 mg/kg ATG-F [28]. Based on these results, ATG-F at a dose of 60 mg/kg or higher has was recommended for matched unrelated donor SCT [20,28]. In the above study ATG was given in 4 equivalent doses from days –5 to –2 or 3 equivalent dosis from days –3 to – 1. It must be noted that of the 31 patients who received ATG \60 mg/kg, 18 (58%) received 20 mg/kg and 11 (35.5%) received 40 mg/kg in 3 or 4 equivalent doses. This split dosing of a relatively low dose of ATG may have led to the poor outcome. In our study, we report better outcome for patients who received 30 mg/kg of ATG-F as a single application on day –1 compared to those receiving 60 mg/kg administered in 3 equivalent doses from day –3 to –1. For the subgroup of patients with CML (n 5 22), our data including patients in both ATG-dose groups are

1,0

0,8

ATG 30 mg/kg

0,6 ATG 60 mg/kg 0,4

0,2

0,0 0

500

1000

1500

2000

2500

3000

3500

days after transplantation Figure 2. Represents Kaplan-Meier estimates and DFS, respectively.

Figure 4. Represents relapse rates obtained using the Gray-test for competing risks.

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comparable with those of the above study with respect to OS (62% versus 66%) and DFS (57% versus 56%). In our study the incidence and severity of aGVHD was similar in the ATG-30 and ATG-60 group. However, there was a trend to increased incidence and severity of cGVHD in patients who received ATG 30 mg/kg (59 versus 40%, P 5 .14). There was no significant difference in the incidence of relapse between the 2 groups. Because more patients in the ATG-30 group died of relapse, we performed analysis using the Graytest taking into consideration competing risks treatment-related mortality and relapse. Treatment-related mortality was higher in the ATG-60 group (12% versus 33%, P 5 .02), mainly because of a higher incidence of fatal infections. Although we do not have data comparing immune reconstitution in the 2 groups, there is substantial evidence that higher doses of ATG are associated with slower immune recovery and a higher incidence of fatal infections [17,24-27]. The lower treatment-related mortality and comparable relapse rate led to a trend to improved DFS in the ATG-30 group (73% versus 51%, P 5 .07). Our results indicate that 30 mg/kg of ATG-F administered as a single dose on day –1 may lead to a better outcome in patients undergoing unrelated donor allogeneic HSCT compared to 60 mg/kg given in 3 equivalent doses. These data are in contrast to the previous report recommending a dose of $60 mg/kg. A prospective randomized study comparing these 2 doses of ATG-F with the discussed timing is warranted and underway. ACKNOWLEDGMENTS We thank the staff of the BMT unit for providing outstanding care to our patients and the medical technicians for their excellent work in the BMT laboratory.

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