Increased Risk for Relapse if ATG is Included in Reduced-Intensity Conditioning HSCT with Sibling Donors

Research Article

Increased Risk for Relapse if ATG is Included in Reduced-Intensity Conditioning HSCT with Sibling Donors

Corresponding author: Dr. Mats Remberger, CAST, Karolinska University Hospital, SE-14186 Stockholm, Sweden, Tel: +46-8-58586391, Email: mats.remberger@ki.se

Abstract

Introduction: The impact of in vivo T-cell depletion on transplantation outcomes in patients treated with reduced-intensity conditioning (RIC) remains controversial.

Patients and Methods: The effect of ATG as part of the Fludarabin + busulphan RIC protocol before sibling donor HSCT was studied. Twenty-one patients were given ATG while 26 did not receive ATG before HSCT. All patients had AML or MDS/MPN, received CsA and MTX as GVHD prophylaxis and received PBSC.

Results: Overall survival at 5 years was 43% in ATG patients and 88% in patients not given ATG (p<0.01). Non-relapse mortality (NRM) was 14% and 4% at one year in the two groups, respectively. The cumulative incidence of relapse at 5 years was 43%  in ATG patients and 15% in patients not receiving ATG (p=0.025). Relapse-free survival (RFS) at 5 years was 38% and 81% (p<0.01), respectively. Cumulative incidences of acute GVHD grades II-IV was 24% and 44% (p=0.16), chronic GVHD was 52% and 78% in the two groups (p=0.08), respectively.

Conclusion: This small study suggests that high dose ATG (8 mg/kg) in sibling donor RIC HSCT increase the risk for relapse and reduce overall survival. Removal of ATG resulted in excellent results.

Keywords: ATG; Relapse; RIC; HSCT

Introduction

Hematopoietic stem cell transplantation (HSCT) is an established curative treatment for patients with hematological malignancies, bone-marrow failure syndrome, primary immunodeficiencies, and inborn metabolic disorders. In the early days of HSCT, most patients were treated with high dose myeloablative chemo/radio-therapy (MAC) with the aim to kill most malignant cells and create space for the new marrow. However, the use of reduced intensity conditioning (RIC) has increased over the last decade. In RIC, lower doses of chemo/ radio-therapy are given with the aim mainly to immune- suppress the patient so the new graft may engraft. Using RIC you mainly rely on the graft-versus-leukemia (GVL) effect to

eradicate malignant cells. The GVL effect is mainly carried out by T-lymphocytes. For this reason removal of T-cells by the use of in vivo T-cell depletion with anti-thymocyte globulin (ATG) may be disadvantageous, as it may increase the risk for reoccurrence of the malignancy.

This hypothesis was verified recently by a large study from CIBMTR. In that study it was shown that relapse incidence increased and relapse-free survival decreased if ATG or alemtuzumab was added to chemotherapy-based RIC in a heterogeneous patient cohort [1]. The present study aimed to investigate these findings in a homogeneous cohort of adult patients with myeloid malignancy given PBSC or BM from HLA-identical siblings after chemotherapy-based RIC.

Patients and Methods

Patients: All consecutive patients with a haematological malignancy treated with hematopoietic stem cell transplantation (HSCT) at Karolinska University Hospital with a sibling donor receiving reduced intensity conditioning (RIC) with fludarabine and busulfan were included. All patients  had acute myeloid leukemia (AML, n=29) or myelodysplastic syndrome/myeloproliferative neoplasm (MDS/MPN, n=18). (Table1).

For disease risk index (DRI) classification, disease in combination with cytogenetic data was used to classify patients into the low, intermediate or high risk patient cohorts [2]. If cytogenetic data was missing or incomplete, patients were assigned to the intermediate disease risk group.

Characteristic Flu+Bu Flu+Bu+ATG p-value
N= 26 21  
Age 61 (43-72) 59 (40-69) 0.90
Sex, M/F 15/11 11/10 0.94
Diagnose:     0.14
AML 19 10  
MDS/MPN 7 11  
DRI (Low/IM/High) 2/19/5 0/15/6 0.51
Donor age 59 (38-71) 56 (46-71) 0.96
Female to Male 6 5 1.0
Stem-cell source

(PBSC)

26 21 1.0
TNC dose (x108/kg) 14.6 (7.2-28.3) 13.6 (6.5-32.7) 0.24
CD34+ cell-dose

(106/kg)

6.8 (2.4-11.1) 7.4 (1.9-14.6) 0.64
CMV serology MM 9 10 0.39
Follow-up (mos) 32 (3-133) 138 (98-164) <0.001

AML; acute myeloid leukemia, MDS/MPN; myelo-dysplastic syndrome/myelo-proliferative neoplasm, DRI; disease risk index, IM; intermediate, Female to Male; female donor to male recipient, PBSC; peripheral-blood stem-cells, TNC dose; total nucleated cell-dose, CMV; cytomegalovirus, MM; mismatch.

Table 1. Characteristics of patients and donors included in the study comparing ATG treatment with no ATG treatment in RIC HSCT.

The study was approved by the Research Ethics Committee of Karolinska Institutet (DNR 425/97). The procedures were in accordance with the Helsinki Declaration.

Donors: All donors were HLA-identical sibling donors.

Conditioning: All patients received fludarabine 30 mg/kg/ day for 6 days and busulfan 4 mg/kg/day for two days [3]. Between 1999 and 2008, ATG was included in the protocol to 21 patients. However, in 2008 ATG was removed for the next 26 patients as we found an increased incidence of relapse. ATG (Thymoglobulin, Genzyme, Cambridge, MN, USA) was given at a dose of 2 mg/kg/day for four days (total dose 8 mg/kg) with the last dose given on the day before HSCT [4].

GVHD prophylaxis: All patients were given orally cyclosporine (CsA) and four doses methotrexate (MTX) as prophylaxis against GVHD. During the first month,  blood  CsA  levels  were kept at 100 ng/mL. In the absence of GVHD CsA was discontinued at three to four months.

Stem cell source: The graft source was peripheral blood stem cells (PBSC) in all cases. Before aphaeresis to obtain PBSC, stem cells were mobilized with subcutaneous G-CSF daily for 4-6 days [5].

Supportive care: Supportive care has been described in detail previously [6,7].

Chimerism analysis: To evaluate lineage-specific chimerism, CD3, CD19 and CD33 –positive cells were selected from peripheral blood using antibody-labeled magnetic beads (Dynal,  Oslo,  Norway).  The  methodology   and   sensitivity of chimerism analysis of the various cell lineages has been described elsewhere [8,9].

Full donor chimerism was defined as more than 95% of donor

cells within the different cell-lineages.

Statistics: Overall survival (OS) was calculated using the Kaplan-Meier method and compared with the log-rank test. Survival time was calculated from the day of transplantation until death or last follow-up. The incidence of transplant- related mortality (TRM), relapse and GVHD were obtained using an estimator of cumulative incidence curves.

Analyses were performed using the cmprsk package (developed by Gray, June 2001), Splus 6.2 and Statistica software.

Results

Survival and non-relapse mortality: Overall survival at 5 years was 43% (95% CI 23-63%) in patients receiving ATG and 88% (76-100%) in patients not given ATG (p<0.01) (Figure 1a). Non-relapse mortality (NRM) was 14% (2-26%) and 4% (0-12%) at one year in the two groups (p=0.14), respectively. Causes of death were relapse (10 and 2), infection (2 and 0), GVHD (1and 1) and secondary malignancy (1 and 0) in patients with and without ATG treatment.

Relapse and relapse-free survival: The cumulative incidence

of relapse at 5 years was 43% (23-63%) in ATG treated patients and 15% (0-31%) in patients not receiving ATG (p=0.025) (Figure 1b). Relapse-free survival (RFS) at 5 years was 38% (18-58%) and 81% (64-98%)(p<0.01) in the two groups, respectively (Figure 1c).

infection occurred in two ATG treated patients but in none of the patients not receiving ATG. No PTLD was seen.

Chimerism: Chimerism was known for 18 patients with ATG

Figure 1. A) Overall survival, B) relapse, C) relapse-free survival and D) chronic GVHD after reduced intensity conditioning and hematopoiet- ic stem cell transplantation with HLA-identical sibling donors, depending on ATG treatment or not.

Graft-versus-host disease: Cumulative incidences of acute GVHD  grades  II-IV  was  24%  (7-41%)  and  44%  (24-64%)

(p=0.16),   grades   III-IV   10%   (0-20%)   and   16% (2-30%)

(p=0.25) in the ATG and non-ATG groups, respectively. The two year OS in patients with grades II-IV acute GVHD was 40% (n=5) and 82% (n=11), p=0.03.

Chronic GVHD was 52% (27-77%) and 78% (61-95%) in the two groups (p=0.08), respectively (Figure 1d). Among the ATG treated patients six had mild, four had moderate and one severe chronic GVHD. In the non-ATG treated patients twelve had mild, four had moderate and two severe chronic GVHD.

Infections: Cytomegalovirus reactivation occurred in 10 and 4 patients in the ATG and non-ATG groups (p=0.025), respectively. There was more herpes-virus infections (CMV, HSV and VZV) in ATG treated patients compared to non-ATG treated patients (67% vs. 23%, p<0.01). Blood stream infection (BSI) occurred in 4 and 2 patients in the two groups (p=0.39). Invasive fungal

and 26 without ATG. Results from the chimerism analysis at different time points are presented in Table 2. Significantly more patients reached full donor chimerism within the CD3+ cell lineages three and six months after HSCT in patients without ATG treatment compared to ATG treated patients.

  1 month 3 month 6 month
  No ATG ATG No ATG ATG No ATG ATG
CD3+ cells 9/26 4/18 21/25 7/18 ** 21/22 10/18 **
CD19+ cells 17/25 10/18 24/25 13/18 # 22/23 15/18
CD33+ cells 12/27 9/18 20/24 12/18 20/23 15/18

#p=0.07, **p≤0.01 vs. no ATG.

Table 2. Numbers of patients who reached full donor chimerism (DC) within the different cell-lineages at different time points after HSCT. Divided into if patients have received ATG or not. (DC/total analyzed patients)

Discussion

In this study we studied the effect of incorporating ATG  in  the Fludarabin and Busulfan RIC protocol before HSCT with sibling donors. The material was homogenous including only patients with myeloid malignancy treated with the Fludarabin and Busulfan protocol, GVHD prophylaxis with cyclosporine and 4 doses of methotrexate. No differences in patient´s and donor´s characteristics existed between the two groups. This is a small study only including 47 patients so the results must be taken with caution. However, we found a negative effect of ATG on OS, RFS and relapse. This is in line with some other studies [1,10,11] while other showed no effect of ATG on OS and relapse [12-16]. The discrepancy between the different studies may probably be an effect of different brands of ATG and different ATG doses. We used a rather high dose (8 mg/kg) compared with many other studies including sibling donor HSCT [13-16], which may have affected the results. In vivo T-cell depletion with ATG removes T-cells from the graft leading to a reduction of the GVL effect. Some studies have showed that this effect probably is dose dependent [4,17]. Using MAC, a strong anti- leukemic effect is performed by the conditioning itself. Using RIC the idea is mainly to immune-suppress the patient by low dose chemo-radiotherapy to ensure engraftment, the anti- leukemic effect is mainly carried out by donor T-cells. For this reason it is logical to presume that it may be more important to spare the GVL effect in RIC HSCT compared to when using MAC. As shown in our study, high dose ATG seems to affect the GVL effect as the incidence of relapse increased. Furthermore, our study showed a statistically non-significant lower incidence of acute and chronic GVHD among ATG treated patients. However, our study was too small to be able to show any statistical difference, but most previous studies have shown that ATG reduce GVHD [4,10,12-15,18,19]. As GVHD carries a GVL effect this is probably one reason for the higher incidence of relapse among the ATG treated patients.

However, the slightly higher incidence of GVHD among the non-ATG patients did not result in higher TRM, as might be feared. The reason for this is that survival among patients with acute GVHD II-IV was significantly better among the non-ATG patients, 82% vs. 40%, p=0.03.

As the non-ATG patients were transplanted in more recent years, this may indicate that we have improved in treatment and care of patients with acute GVHD. This has been reported previously by our group [20].

Viral infections were more common in ATG treated patients. This has been proposed in previous studies [1,4,21]. By removing T-cells from the infused graft the defence against infections will be reduced.

This small but homogenous study shows that high dose ATG is not indicated in RIC HSCT using sibling donors. However, we cannot exclude that some subgroups of sibling donor HSCT

patients may benefit from low dose ATG, eg. patients with high-risk features to develop severe acute or chronic GVHD. This has to be studied in larger randomized studies.

Acknowledgments

 

This study was supported by grants from the Swedish Cancer Society (CF2014-2016), the Swedish Children’s Cancer Foundation (PR2013-0022 and KF2013-0011), the Marianne and Marcus Wallenbergs Foundation (2013.0117) and grants provided by the Stockholm County Council (ALF-project 20140451).

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